Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-11-26 to 2014-12-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
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)
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
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 003
- Expiration date of the lot/batch: 01 Jan 2020

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, exclusion of direct sunlight, test substance is hygroscopic
- Stability under test conditions: stable

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (phenobarbital and β-naphthoflavone induced rat liver)
Test concentrations with justification for top dose:
0; 33; 100; 333; 1 000; 2 700 and 5400 μg/plate
Vehicle / solvent:
- Vehicle used: DMSO
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
2-AA
Positive control substance:
other: 2-Aminoanthracene
Remarks:
with S9 mix (all tester strains tested)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
MNNG
Positive control substance:
other: N-methyl-N'-nitro-N-nitrosoguanidine
Remarks:
without S9 mix; 5 µg/plate (TA1535, TA100)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
NOPD
Positive control substance:
other: 4-nitro-o-phenylenediamine
Remarks:
without S9 mix; 10 μg/plate (TA 98)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
AAC
Positive control substance:
9-aminoacridine
Remarks:
without S9 mix; 100 µg/plate (TA1537)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
4-NQO
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9 mix; 5 µg/plate (E. coli WP2 uvrA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) (SPT); preincubation (PIT)

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 - 72 hours in the dark at 37 °C

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of his+ or trp+ revertants

Evaluation criteria:
Acceptance criteria
Generally, the experiment was considered valid if the following criteria were met:
• The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
• The sterility controls revealed no indication of bacterial contamination.
• The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
• Fresh bacterial culture containing approximately 10^9 cells per mL were used.

Assessment criteria
The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA98, TA100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA1535 and TA1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.

A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control data range under all experimental conditions in at least two experiments carried out independently of each other.

Results and discussion

Test results
Key result
Species / strain:
bacteria, other: TA98, TA100, TA1535, TA1537, E. coli WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Evaporation from medium: No
- Water solubility: No
- Precipitation:

RANGE-FINDING/SCREENING STUDIES:

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
- Negative (solvent/vehicle) historical control data:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: [complete, e.g. CBPI or RI in the case of the cytokinesis-block method; RICC, RPD or PI when cytokinesis block is not used]
- Other observations when applicable: [complete, e.g. confluency, apoptosis, necrosis, metaphase counting, frequency of binucleated cells]

Any other information on results incl. tables

Table 1 Raw data

Experiment 1 (SPT)

Dose (µg/plate)

Mean number of revertant colonies/3 replicate plates with different strains of Salmonella typhimurium and Escherichia coli

 

TA1535

TA1537

TA98

TA100

WP2 uvrA

 

 

 

Results with S9

DMSO

12

9.7

31

37

66.7

33

11.7

9.3

27.7

32.3

66.7

100

9.3

8.7

33.7

38

63

333

12.7

8.7

30.3

37.7

64.7

1000

11.3

8

26.7

29

71.3

2700

11.7

3

30.7

43.7

60.3

5400

11

4

22

34.3

52.7

positive control

202

140.3

1672.7

746.3

206

 

 

 

Results without S9

DMSO

11.7

7.3

25

38.7

65

33

10.3

7.3

25

35.7

60.3

100

12.3

10

23

40.3

61.3

333

12

7.3

22.7

31.3

59.7

1000

12

11

27.3

34

47

2700

15

6.3

20.7

42.3

48

5400

10.7

1.7

20.3

24.3

38.7

positive control

4552

697.7

444

4474

900.3

 

 

 

 

 

 

Experiment 2 (PIT)

Dose (µg/plate)

Mean number of revertant colonies/3 replicate plates with different strains of Salmonella typhimurium and Escherichia coli

 

TA1535

TA1537

TA98

TA100

WP2 uvrA

 

 

 

Results with S9

DMSO

9

10.3

28.3

28.3

86.7

33

9.3

7.3

32.3

29.7

88

100

9.3

7.3

30

27.3

86.3

333

9.7

6.7

25.7

29

94.3

1000

7.7

6.3

35

30.7

88.3

2700

9

5.7

19

27.3

97.7

5400

7.3

6

21

29.7

79

positive control

179.3

116.7

1276.3

663

208.7

 

 

 

Results without S9

DMSO

11.3

7.3

21

31.7

99.7

33

9.7

7.7

20.3

27

90.3

100

12

5.3

19.7

28.7

97

333

8

4.3

22.3

25.3

79.7

1000

9.3

8.3

16

23.3

92

2700

8.7

4.3

21

24.3

75.7

5400

7

4.7

19.3

29

89.7

positive control

1568.3

591.3

354.3

2092.3

253

 

Applicant's summary and conclusion