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 -01-19 till 2016-02-18
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
Report Date:
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

Method

Species / strain
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-Naphthoflavone induced rat liver S9 (experiment I) and non-induced hamster liver S9 (experiment II)
Test concentrations with justification for top dose:
3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate / pre-experiment/experiment I
1; 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate / experiment II
Vehicle:
- Vehicle(s)/solvent(s) used: deionized water
- Justification for choice of solvent/vehicle: best suitable solvent
Controls
Negative controls:
yes
Solvent 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; Congo red
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation;


DURATION
- Preincubation period: 30 minutes
- 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 valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other:
Remarks:
Migrated from field 'Test system'.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS

- Water solubility: soluble
- Precipitation: The test item precipitated in the overlay agar in the test tubes from 10 to 5000 µg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 100 to 5000 µg/plate. The undissolved particles had no influence on the data recording.
- Other confounding effects: none
COMPARISON WITH HISTORICAL CONTROL DATA: In experiment II, the data in solvent controls of strain TA 100 without S9 and strain 1535 with S9 mix and the untreated controls of strains TA 1537 and TA 100 with S9mix were slightly above our historical control range. Since this deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies and had no detrimental impact on the outcome of the study.
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 all strains with and without metabol¬ic activation. Only in experiment II minor toxic effects were observed in strain TA 1535 with S9 mix at 5000 µg/plate.

Any other information on results incl. tables

Summary Tabellen

Table1            Summary of Experiment I

Study Name: 1743802

Study Code: Envigo 1743802

Experiment: 1743802 VV Plate

Date Plated: 19/01/2016

Assay Conditions:

Date Counted: 22/01/2016

 

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

Deionised water

 

 

11 ± 5

9 ± 4

25 ± 8

181 ± 30

45 ± 9

Untreated

 

 

8 ± 1

13 ± 4

26 ± 10

199 ± 7

49 ± 5

Aluminium-

3 µg

 

11 ± 5

10 ± 4

29 ± 5

185 ± 22

45 ± 10

Braun 1134 p

10 µg

 

12 ± 4

10 ± 4

21 ± 1

172 ± 12

50 ± 17

 

33 µg

 

10 ± 3

10 ± 4

25 ± 5

179 ± 5

51 ± 7

 

100 µg

 

10 ± 2P

9 ± 2P

26 ± 6P

174 ± 13P

48 ± 5P

 

333 µg

 

10 ± 4P

8 ± 2P

28 ± 2P

196 ± 19P

44 ± 12P

 

1000 µg

 

9 ± 4P

6 ± 1P

27 ± 8P

194 ± 18P

40 ± 6P

 

2500 µg

 

12 ± 2P

7 ± 2P

34 ± 6P

185 ± 18P

40 ± 6P

 

5000 µg

 

9 ± 4P

8 ± 2P

27 ± 4P

172 ± 11P

31 ± 3P

NaN3

10 µg

 

1171 ± 88

 

 

2083 ± 137

 

4-NOPD

10 µg

 

 

 

302 ± 36

 

 

4-NOPD

50 µg

 

 

63 ± 5

 

 

 

MMS

2.0 µL

 

 

 

 

 

999 ± 76

 

 

 

 

 

 

 

 

 

With Activation

Deionised water

 

 

13 ± 3

12 ± 2

30 ± 5

185 ± 17

45 ± 4

Untreated

 

 

10 ± 3

10 ± 3

28 ± 6

195 ± 29

59 ± 8

Aluminium-

3 µg

 

13 ± 5

10 ± 5

28 ± 2

189 ± 15

51 ± 8

Braun 1134 p

10 µg

 

12 ± 5

7 ± 2

25 ± 13

173 ± 25

52 ± 6

 

33 µg

 

13 ± 3

8 ± 2

27 ± 6

186 ± 21

52 ± 10

 

100 µg

 

10 ± 4P

9 ± 3P

24 ± 5P

191 ± 5P

62 ± 8P

 

333 µg

 

11 ± 4P

11 ± 5P

31 ± 5P

202 ± 17P

50 ± 3P

 

1000 µg

 

7 ± 1P

7 ± 3P

27 ± 4P

200 ± 23P

58 ± 9P

 

2500 µg

 

9 ± 1P

10 ± 5P

30 ± 4P

176 ± 27P

43 ± 3P

 

5000 µg

 

10 ± 5P

10 ± 3P

26 ± 6P

147 ± 25P

31 ± 7P

2-AA

2.5 µg

 

510 ± 32

107 ± 11

4431 ± 85

4790 ± 229

 

2-AA

10.0 µg

 

 

 

 

 

359 ± 14

 

 

 

 

 

 

 

 

 

 

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

Precipitate

 

 

Table2            Summary of Experiment II

Study Name: 1743802

Study Code: Envigo 1743802

Experiment: 1743802 HV2 Pre

Date Plated: 12/02/2016

Assay Conditions:

Date Counted: 18/02/2016

 

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

Deionised water

 

 

14 ± 5

8 ± 1

33 ± 8

197 ± 5

52 ± 10

Untreated

 

 

16 ± 5

7 ± 1

30 ± 4

206 ± 6

55 ± 5

Aluminium-

1 µg

 

14 ± 2

8 ± 1

33 ± 9

247 ± 6

55 ± 7

Braun 1134 p

3 µg

 

15 ± 5

9 ± 2

32 ± 3

259 ± 7

59 ± 7

 

10 µg

 

13 ± 2

9 ± 3

29 ± 7

263 ± 24

49 ± 10

 

33 µg

 

13 ± 4

9 ± 1

34 ± 10

267 ± 14

50 ± 8

 

100 µg

 

14 ± 5P

9 ± 1P

33 ± 4P

253 ± 35P

51 ± 8P

 

333 µg

 

13 ± 3P

8 ± 3P

29 ± 7P

271 ± 25P

47 ± 2P

 

1000 µg

 

16 ± 4P

15 ± 1P

29 ± 2P

249 ± 25P

50 ± 7P

 

2500 µg

 

12 ± 4P

8 ± 1P

36 ± 6P

252 ± 22P

56 ± 2P

 

5000 µg

 

9 ± 4P

8 ± 1P

49 ± 6P

246 ± 9P

39 ± 11P

NaN3

10 µg

 

1421 ± 64

 

 

2394 ± 58

 

4-NOPD

10 µg

 

 

 

371 ± 10

 

 

4-NOPD

50 µg

 

 

72 ± 13

 

 

 

MMS

2 µL

 

 

 

 

 

1169 ± 126

 

 

 

 

 

 

 

 

 

With Activation

Deionised water

 

 

22 ± 3

27 ± 4

49 ± 3

204 ± 4

55 ± 11

Untreated

 

 

25 ± 4

27 ± 4

55 ± 1

216 ± 16

61 ± 2

Aluminium-

1 µg

 

26 ± 3

26 ± 3

51 ± 2

225 ± 18

60 ± 12

Braun 1134 p

3 µg

 

28 ± 2

32 ± 3

53 ± 2

230 ± 23

54 ± 11

 

10 µg

 

28 ± 2

32 ± 1

56 ± 1

235 ± 25

56 ± 6

 

33 µg

 

27 ± 5

30 ± 3

58 ± 11

242 ± 3

55 ± 1

 

100 µg

 

25 ± 6P

31 ± 3P

54 ± 2P

233 ± 5P

64 ± 9P

 

333 µg

 

21 ± 3P

32 ± 4P

51 ± 10P

240 ± 9P

63 ± 6P

 

1000 µg

 

22 ± 1P

25 ± 3P

59 ± 11P

250 ± 7P

53 ± 4P

 

2500 µg

 

14 ± 4P

20 ± 3P

81 ± 7P

231 ± 19P

47 ± 2P

 

5000 µg

 

9 ± 1P

20 ± 4P

82 ± 6P

242 ± 48P

49 ± 13P

2-AA

2.5 µg

 

 

 

 

2251 ± 49

 

2-AA

2.5 µg

 

395 ± 21

119 ± 12

 

 

 

2-AA

10 µg

 

 

 

 

 

832 ± 103

Congo red

500 µg

 

 

 

328 ± 11

 

 

 

 

 

 

 

 

 

 

 

 

Key to Positive Controls

Key to Plate Postfix Codes

 

 

NaN3

2-AA

4-NOPD

Congo red

MMS

sodium azide

2-aminoanthracene

4-nitro-o-phenylene-diamine

Congo red

methyl methane sulfonate

P

Precipitate

 

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with and without rat- and hamster S9

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:

This study was performed to investigate the potential of the test substance to induce gene mutations according to the plate incorporation test (experiment I) with and without rat S9 mix and the pre-incubation test (experiment II) with and without hamster S9 mix using theSalmonella typhimuriumstrains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia colistrain WP2 uvrA.

The assay was performed in two independent experiments both 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:                                1; 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The test item precipitated in the overlay agar in the test tubes from 10 to 5000 µg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 100 to 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 all strains with and without metabolic activation. Only in experiment II minor toxic effects were observed in strain TA 1535 with S9 mix at 5000 µg/plate. No substantial increase in revertant colony numbers of any of the fivetester strains was observed following treatment with Aluminium-Braun 1134 p at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.