Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 Aug - 01 Sep 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
1993
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

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

Method

Target gene:
his operon, trp operon
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9 induced by ß-Naphthoflavone/phenobarbital
Test concentrations with justification for top dose:
1st series: 5, 15.8, 50, 158, 500, 1580, 5000 µg/plate (with and without S9 mix)
2nd series: 158, 500, 1580, 2810, 5000 µg/plate (with and without S9 mix)
Vehicle / solvent:
- Solvent used: ultra pure water, final concentration 100 µL per plate
- Justification for choice of solvent: solubitlity properties of the test item, non toxicity to bacteria
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
sodium azide
other: 2-aminoanthracene 2-10 µg/plate, daunomycin 1 µg/plate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 2-3 days

NUMBER OF REPLICATIONS: 3 replicates for test item concentrations and positive controls, 6 replicates for solvent controls

DETERMINATION OF CYTOTOXICITY
- Method: counting numbers of revertants

OTHER:
-S9 concentration: 1st series 10%, 2nd series 20%

Evaluation criteria:
A test material was to be defined as positive or mutagenic in this assay if
- the assay is considered valid AND
- a biologically relevant increase in the mean number of revertants above a threshold of 2-fold (TA98, TA100, WP2 uvrA) or 3-fold (TA1535, TA1537) as compared to the concurrent negative controls is observed.
- an increase exceeding the threshold at only one concentration is considered as biologically meaningful if reproduced in a 2nd independent experiment.
- a concentration-dependent increase is considered biologically meaningful if the threshold is exceeded at more than one concentration

A test material is considered negative or non-mutagenic in this assay if
- the assay is considered valid AND
- none of the above criteria are met
Statistics:
Not performed as not mandatory for this test system.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
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:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
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:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
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:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
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:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with
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:
not examined
Positive controls validity:
valid
Additional information on results:
Following treatment with the test item, no precipitation on the agar plates occurred. No toxicity to the bacteria was observed.
Under the experimental conditions described, the test item induced relevant, concentration-dependent increases in revertant numbers exceeding a threshold of 2-fold (TA98, TA100, WP2 uvrA) or 3-fold (TA1535, TA1537) of the negative control value in all tester strains in the absence of S9-mix with the exception of TA1535. In the presence of S9-mix there was a relevant increase present in TA1537 only.
According to the criteria for negative and positive results as predetermined in the study plan, the test item was clearly mutagenic under the described experimental conditions.

Any other information on results incl. tables

Table 1: Summary of Experiment 1

Metabolic Activation

Test material

Concentr. (µg/plate)

Revertant Colony Counts (Mean ± SD)

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

Without Activation

H20

 

34 +/- 11

124 +/- 5

29 +/- 6

9 +/- 4

33 +/- 8

Test item

5

30 +/- 10

129 +/- 9

21 +/- 2

8 +/- 3

39 +/- 6

15.8

38 +/- 12

139 +/- 23

25 +/- 6

10 +/- 6

37 +/- 6

50

37 +/- 8

140 +/- 19

26 +/- 5

14 +/- 1

37 +/- 2

158

40 +/- 7

168 +/- 16

18 +/- 1

27 +/- 4

44 +/- 7

500

42 +/- 1

223 +/- 18

20 +/- 4

71 +/- 5

75 +/- 9

1580

52 +/- 13

444 +/- 3

38 +/- 7

187 +/- 10

184 +/- 17

5000

112 +/- 16

1435 +/- 126

49 +/- 10

506 +/- 9

605 +/- 19

DAUN

1

166 +/- 26

 

 

 

 

NaN3

2

 

1365 +/- 14

787 +/-/ 13

 

 

9-AA

50

 

 

 

1153 +/- 266

 

NQO

2

 

 

 

 

2101 +/- 163

With Activation

H20

 

46 +/- 3

125 +/- 12

17 +/- 6

10 +/- 5

41 +/- 4

Test item

5

35 +/- 6

119 +/- 15

22 +/- 1

10 +/- 6

43 +/- 11

15.8

37 +/- 8

119 +/- 14

19 +/- 3

10 +/- 4

37 +/- 6

50

43 +/- 5

133 +/- 18

22 +/- 3

15 +/- 3

41 +/- 2

158

42 +/- 15

141 +/- 7

22 +/- 2

19 +/- 4

32 +/- 6

500

37 +/- 12

150 +/- 9

16 +/- 6

25 +/- 3

41 +/- 6

1580

39 +/- 5

168 +/- 11

22 +/- 2

67 +/- 4

54 +/- 5

5000

41 +/- 2

218 +/- 33

22 +/- 9

173 +/- 5

58 +/- 6

2-AA

2

614 +/- 17

1237 +/- 276

 

 

2-AA

5

 

 

209 +/- 12

275 +/- 27

 

2-AA

10

 

 

 

 

320 +/- 18

Table 1: Summary of Experiment 2

Metabolic Activation

Test material

Concentr. (µg/plate)

Revertant Colony Counts (Mean ± SD)

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

Without Activation

H20

 

31 +/- 10

132 +/- 19

27 +/- 7

8 +/- 3

31 +/- 7

Test item

5

--

--

--

--

--

15.8

--

--

--

--

--

158

32 +/- 10

161 +/- 4

23 +/- 3

34 +/- 5

50 +/- 6

500

32 +/- 4

240 +/- 10

24 +/- 7

74 +/- 3

105 +/- 18

1582

55 +/- 11

484 +/- 9

30 +/- 9

232 +/- 31

242 +/- 8

2810

81 +/- 5

845 +/- 38

30 +/- 4

360 +/- 16

401 +/- 24

5000

106 +/- 22

1576 +/- 31

51 +/- 3

576 +/- 31

688 +/- 12

DAUN

1

192 +/- 45

 

 

 

 

NaN3

2

 

1754 +/- 45

887 +/- 62

 

 

9-AA

50

 

 

 

580 +/- 44

 

NQO

2

 

 

 

 

2170 +/- 127

With Activation

H20

 

41 +/- 7

150 +/- 14

15 +/- 6

12 +/- 3

42 +/- 6

Test item

5

--

--

--

--

--

15.8

--

--

--

--

--

158

42 +/- 1

145 +/- 7

18 +/- 6

13 +/- 3

34 +/- 6

500

33 +/- 4

135 +/- 10

14 +/- 2

19 +/- 9

41 +/- 1

1580

27 +/- 11

158 +/- 8

10 +/- 3

40 +/- 14

52 +/- 4

2810

32 +/- 9

176 +/- 23

11 +/- 3

56 +/- 9

53 +/- 8

5000

28 +/- 5

202 +/- 17

8 +/- 1

86 +/- 13

72 +/- 8

2-AA

2

183 +/- 2

815 +/- 59

 

 

2-AA

5

 

 

80 +/- 5

127 +/- 9

 

2-AA

10

 

 

 

 

221 +/- 14

Key to Positive Controls

NaN3                Sodium azide

2-AA                2-Aminoanthracene

9-AA                9-Aminoacridine

DAUN              Daunomycin

NQO                4-Nitroquinoline-N-oxide

Applicant's summary and conclusion

Conclusions:
The test item is considered mutagenic in bacteria under the test conditions described.