Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 Nov 2017 - 9 Feb 2018
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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 21 Jul 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
30 May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
Aug 1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
GLP-Landesleitstelle Bayern, Bayerisches Landesamt für Gesundheit, Schwabach, Germany
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/β-naphthoflavone
Test concentrations with justification for top dose:
Pre-experiment: 3.16, 10, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate with and without metabolic activation for TA 98 and TA 100

Experiment: 31.6, 100, 316, 1000, 2500 and 5000 µg/plate with and without metabolic activation

The test substance concentrations used in the main experiment were chosen according to the results of the pre-experiment.
Vehicle / solvent:
- Vehicle/solvent used: Distilled water
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 acitivity.
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 (4-NOPD), 2-aminoanthracene (2-AA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) (pre-experiment and main experiment 1) and pre-incubation test (experiment 2)

DURATION
- Preincubation period: 1 h
- Exposure duration: at least 48 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants down to a mutation factor of approximately ≤ 0.5 or a clearing of the bacterial background lawn

POSITIVE CONTROL 2-AA:
- Proficiency of the activity of S9 was determined and confirmed prior to the experiment
Evaluation criteria:
The mutation factor is calculated by dividing the mean value of the revertant count by the mean values of the solvent control (the exact and not rounded values are used for calculation)
A test item is considered mutagenic if:
- a clear dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabloic activation.

A biologically relevant increase is describes as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.

A test item producing neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.
Statistics:
Mean values and standard deviations were calculated.

Results and discussion

Test results
Key result
Species / strain:
other: S. typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 102
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
- Precipitation: No precipitation of the test item occurred up to the highest investigated dose with and without metabolic activation.

RANGE-FINDING/SCREENING STUDIES: Only strain TA 98 and TA 100 were tested in the pre-experiment.

ADDITIONAL INFORMATION ON CYTOTOXICITY: No toxic effects of the test item was observed in any strain.

Any other information on results incl. tables

Table 1: Summary of Pre-Experiment

Substance

Dose

(µg/plate)

TA98

Mutation Factor (toxicity*)

TA 100

Mutation Factor (toxicity*)

 

without S9

with S9

without S9

with S9

Solvent Control

(Distilled water)

1.0

1.0

1.0

1.0

4-NOPD

10.0

12.4

-

-

-

NaN3

10.0

-

-

6.7

-

2-AA

2.50

-

58.7

-

13.8

 

Test Item

3.16

0.8

0.9

0.9

0.9

10.0

0.9

0.7

0.9

1.0

31.6

0.8

0.9

0.9

0.9

100

0.8

0.8

0.9

0.9

316

0.8

0.6

0.8

0.8

1000

0.8

0.8

0.8

1.0

2500

0.9

1.0

1.0

1.0

5000

0.9

0.7

1.1

1.1

* toxicity parameter: B = Background lawn reduced, N = No background lawn

 

Table 2: Experiment 1 (Plate-incorporation Test)

 

EXPERIMENT 1 (Revertant colonies per plate ± SD)

 

S9-Mix

Without

 

 

Test item (µg/plate)

TA 98

TA 100

TA 1535

TA 1537

TA 102

Distilled water

 

42 ± 0.6

94 ± 7.0

22±10.3

22±2.1

284±17.6

Test Item

31.6

35 ± 1.7

86 ± 10.0

24±3.6

31±5.0

275±28.0

100

33 ± 7.1

84 ± 1.2

28±2.5

22±7.0

261±14.0

316

35 ± 7.1

79 ± 6.4

23±2.6

28±2.6

268±35.7

1000

35 ± 6.0

74 ± 4.6

21±6.7

20±1.2

280±23.9

2500

39 ± 9.2

93 ± 3.5

16±4.2

23±3.1

265±13.2

5000

37 ± 7.2

101 ± 12.7

18±4.0

24±2.5

257±55.4

4-NOPD

10 (TA 98)/40 (TA 1537)

524 ± 32.9

-

-

234±10.3

-

NaN3

10

-

631 ± 41.1

1155±2.9

-

-

MMS

1

 

 

 

 

1915±320.4

 

S9-Mix

With

 

 

Test item (µg/plate)

TA 98

TA 100

TA 1535

TA 1537

TA 102

Distilled water

-

34 ± 4.4

89±9.5

14±1.5

27±4.0

318±25.4

 

31.6

31 ± 6.7

80±4.6

19±1.2

31±6.1

320±12.7

 

100

26 ± 7.8

76±5.3

21±1.0

30±4.6

330±45.6

 

316

22 ± 2.0

71±1.5

17±3.6

26±8.7

320±13.1

 

1000

26 ± 5.9

87±25.4

14±7.0

23±2.5

368±34.1

 

2500

35 ± 4.4

85±21.2

18±12.5

21±1.0

373±18.0

 

5000

25 ± 2.3

95±2.0

13±4.7

24±3.2

348±27.6

2-AA

2.5/

10 (only TA 102)

1994 ± 258.6

1225±45.7

268±10.1

225±58.0

955±151.7

 

NC = Negative/Vehicle Control

PC = Respective positive control substances (for details see method description)

SD = Standard Deviation

Table 3: Experiment 2 (Pre-incubation Test)

 

EXPERIMENT 2 (Revertant colonies per plate ± SD)

 

S9-Mix

Without

 

 

Test item (µg/plate)

TA 98

TA 100

TA 1535

TA 1537

TA 102

Distilled water

 

20 ± 3.1

128 ± 6.4

18 ± 3.0

21 ± 3.1

250 ± 35.4

Test Item

31.6

19 ± 4.0

105 ± 6.1

19 ± 2.0

21 ± 2.1

267 ± 38.8

100

18 ± 3.5

114 ± 9.1

16 ± 1.5

20 ± 3.0

207 ± 25.5

316

19 ± 6.6

115 ± 9.0

19 ± 1.5

20 ± 1.0

184 ± 16.8

1000

19 ± 4.6

115 ± 18.2

20 ± 2.5

24 ± 0.6

213 ± 1.0

2500

18 ± 2.1

114 ± 27.1

17 ± 2.1

22 ± 1.5

234 ± 5.9

5000

20 ± 3.1

119 ± 23.4

20 ±.5

21 ± 2.6

219 ± 29.2

4-NOPD

10 (TA 98)/40 (TA 1537)

342 ± 27.6

-

-

115 ± 15.6

-

NaN3

10

-

590 ± 66.5

268 ± 116.3

-

-

MMS

1

-

-

-

-

1296 ± 213.2

 

S9-Mix

With

 

 

Test item (µg/plate)

TA 98

TA 100

TA 1535

TA 1537

TA 102

Distilled water

-

18 ± 4.5

117 ± 23.1

14 ± 3.6

17 ± 2.6

220 ± 40.8

 

31.6

20 ± 3.5

100 ± 12.7

15 ± 2.5

18 ± 3.1

239 ± 1.7

 

100

18 ± 2.1

99 ± 21.0

13 ± 1.5

18 ± 1.5

240 ± 4.6

 

316

19 ± 6.4

100 ± 16.3

15 ± 0.6

20 ± 1.0

239 ± 0.0

 

1000

17 ± 1.7

101 ± 20.3

15 ± 1.5

19 ± 2.5

241 ± 3.1

 

2500

21 ± 2.3

104 ± 21.0

14 ± 2.0

21 ± 3.8

255 ± 5.1

 

5000

18 ± 2.5

109 ± 13.4

15 ± 2.0

19 ± 2.1

262 ± 1.2

2-AA

2.5/

10 (only TA 102)

215 ± 9.7

420 ± 0.6

122 ± 21.9

151 ± 20.3

838 ± 23.8

 

NC = Negative/Vehicle Control

PC = Respective positive control substances (for details see method description)

SD = Standard Deviation

Applicant's summary and conclusion

Conclusions:
Under the conditions of the conducted test the item was not mutagenic in any of the five strains (TA 98, TA 100, TA 1535, TA 1537 and TA 102) tested with and without metabolic activation up to 5000 µg/plate.