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:
2013-07-19 to 2013-08-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study according to OECD and EU guideline and in accordance with GLP
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report):FC-131
- Physical state: yellow crystalline powder
- Analytical purity: 99.8% (area HPLC)
- Impurities (identity and concentrations): about 0.1% 4-diethylamino-2-methoxy-benzaldehyde
- Isomers composition:No data
- Purity test date:03.07.2013
- Lot/batch No.:30610045
- Expiration date of the lot/batch:12.06.2015
- Homogeneity: yes
- Storage condition of test material: Room temperature (20+-5 °C), no humidity
- Production date: Jun. 2013

Method

Target gene:
missing Histidine synthesis
Species / strain
Species / strain:
other: Salmonella typhimurium LT2: TA1535, TA 97a, TA98, TA 100, TA 102
Details on mammalian cell lines (if applicable):
- Type and identity of media: Nutrient broth for overnight culture (Merck 5443)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data
- Genotype confirmation was performed once a quarter
- Periodically checked for karyotype stability: yes/no
- Periodically "cleansed" against high spontaneous background: yes/no
Additional strain characteristics:
other: all with mutation in histidine gene
Metabolic activation:
with and without
Metabolic activation system:
produced from the livers of male Sprague-Dawley rats treated with 500 mg Aroclor 1254/kg b.w. intraperitoneally
Test concentrations with justification for top dose:
nominal concentrations:
- first experiment (plate incorporation method):
5002 µg/plate , 1501 µg/plate, 502 µg/plate, 152 µg/plate and 51 µg/plate (= 50.02; 15.01 ;5.02; 15.2 and 0.51 g/L)
- second experiment (pre-incubation method):
5002 µg/plate , 2500 µg/plate, 1254 µg/plate, 624 µg/plate, 313 µg/plate, 157 µg/plate and 76 µg/plate (= 50.02; 25 ; 12.54; 6.24; 3.13; 1.57 and 0.76 g/L)
Vehicle:
- Vehicle(s)/solvent(s) used:DMSO

- Justification for choice of solvent/vehicle:
The test item was not sufficiently soluble in deionised water, dimethyl sulfoxide (DMSO) or ethanol. In DMSO, the test item showed the best homogenous suspension under permanent shaking in the highest required concentration.
Therefore, the test item was weighed directly. The weighed amounts were autoclaved and filled up with sterile DMSO.
The suspensions were shaken for 24 hours and stirred during application.
In both experiments, the two highest concentrations were tested as suspension (in the first experiment: 5002 µg/plate and 1501 µg/plate; in the second experiment: 5002 µg/plate and 2500 µg/plate).
Complete dissolution was observed at the following nominal concentrations:
Experiment I: 502 µg/plate, 152 µg/plate and 51 µg/plate.
Experiment II: 1254 µg/plate, 624 µg/plate, 313 µg/plate, 157 µg/plate and 76 µg/plate.
Controls
Negative controls:
yes
Remarks:
H2O
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
other: 4-Nitro- 1,2-phenylene diamine, 2-Amino-anthracene
Remarks:
NPD (TA97a, TA98, TA102; no metabolic activation); Na-azide (TA 100, TA 1535, no metabolic activation), 2AA (TA 97a, TA100, TA 102, TA 1535, +S9), BaP (TA 98, + S9)
Details on test system and conditions:
METHOD OF APPLICATION:
experiment 1: in agar (plate incorporation);
experiment 2: preincubation

DURATION:
- Preincubation period: 20min
- Exposure duration:48h (second experiment no data)
- Expression time (cells in growth medium): at least 1E+09 cells/ml


NUMBER OF REPLICATIONS:
4 plates for each concentration, strain, metabolic activation/no metabolic activation

NUMBER OF CELLS EVALUATED: see Table 1a (experiment 1) and Table 1b (experiment 2) below

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (The test item is considered non-toxic, if the quotient titre/tox is below 2.)

Evaluation criteria:
The colonies were counted visually, the numbers were recorded. A spreadsheet software (Microsoft Excel®) was used to calculate mean values and standard deviations of each treatment, solvent control and positive control.
The increase factor f(I) of revertant induction (mean revertants divided by mean spontaneous revertants) and the absolute number of revertants (“Rev. abs.”, mean revertants less mean spontaneous revertants) were also calculated.
A test item is considered to have mutagenic potential, if a significant, reproducible increase of revertant colonies per plate (increase factor 2) in at least one strain can be observed. A concentration-related increase over the range tested can also be taken as a sign of mutagenic activity.
All calculations are performed with unrounded values.
Statistics:
calculation of mean values and standard deviations, calculation of increase factor, no documentation of significance determination

Results and discussion

Test results
Species / strain:
other: TA1535, TA97a, TA98, TA100 and TA102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: test item not completely soluble in DMSO (undissolved particles have been observed) at the two highest tested concentrations (5002 and 1501 µg/plate) and were tested as suspensions in these concentrations
- Precipitation: undissolved substance particles have been observed at the highest tested concentrations


RANGE-FINDING/SCREENING STUDIES: no data

COMPARISON WITH HISTORICAL CONTROL DATA: only marginal differences compared to historical cotrol data

OTHER CONTROL DATA:

Genotype Confirmation: ok
Histidine Requirement: ok
UV-Sensitivity (uvrB): ok
Crystal Violet Sensitivity (deep rough/rfa): ok

Any other information on results incl. tables

Table 3: Results of experiment 1 (negative controls, positive controls, values for FC-131)

Strain

TA97a

TA98

TA100

TA102

TA1535

Induction

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

H2O

Mean

150

125

15

15

110

123

180

184

22

22

sd

9.9

16.4

5.9

3.6

5.1

13.6

25.2

15.2

4.4

2.2

DMSO

Mean

126

103

16

14

113

140

174

169

22

20

sd

12.3

7.9

5.4

2.6

6.9

13.9

18.2

23.9

5.5

6.2

Positive
Controls*

Mean

600

552

139

110

619

520

683

895

468

112

sd

81

84

17

14

55

84

54

76

39

17

f(I)

4.76

5.36

8.69

7.86

5.63

3.71

3.93

5.30

21.27

5.60

5002 µg/pl.

Mean

107

109

18

14

128

116

179

196

20

21

sd

7

16

1

2

19

18

13

12

3

3

f(I)

0.85

1.06

1.13

1.00

1.13

0.83

1.03

1.16

0.91

1.05

1501 µg/pl.

Mean

112

102

16

18

109

131

190

175

22

20

sd

8

5

3

2

18

25

21

15

3

3

f(I)

0.89

0.99

1.00

1.29

0.96

0.94

1.09

1.04

1.00

1.00

502 µg/pl.

Mean

120

121

13

13

119

136

167

178

21

23

sd

9

20

3

2

20

20

15

19

7

4

f(I)

0.95

1.17

0.81

0.93

1.05

0.97

0.96

1.05

0.95

1.15

152 µg/pl.

Mean

127

120

16

13

122

122

185

189

22

24

sd

13

26

3

2

7

7

14

17

2

2

f(I)

1.01

1.17

1.00

0.93

1.08

0.87

1.06

1.12

1.00

1.20

51 µg/pl.

Mean

112

116

13

13

116

122

193

192

24

23

sd

21

9

3

1

19

3

19

18

3

3

f(I)

0.89

1.13

0.81

0.93

1.03

0.87

1.11

1.14

1.09

1.15

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative without metabolic activation
negative with metabolic activation

Under the test conditions, the test item FC-131 is not mutagenic in the Bacterial Reverse Mutation Test using Salmonella typhimurium, strains TA 97a, TA 98, TA 100, TA 102 and TA 1535 with or without metabolic activation. Cytotoxicity of the test item at the concentrations tested was not detected.
No complete dissolution in DMSO/ Ethanol/water was possible for the two highest concentrations in the first and in the second experiment; hence undissolved particles were visible on the plates and substance was tested as suspension.
Executive summary:

The mutagenic potential of FC-131 was tested according to the OECD guideline 471 with the Bacterial Reverse Mutation Test using five strains of Salmonella typhimurium TA 97a, TA 98, TA 100, TA 102 and TA 1535 with and without metabolic activation (using S9) and applying both the plate incorporation and pre-incubation method. The tests and all included controls were valid. FC-131 was tested with a concentration range between 76 µg/plate (= 0.76 g/L) and 5002 µg/plate (= 50 g/L). Concentrations above 1501 µg/plate showed precipitation. No cytotoxicity was observed upto and including the highest concentration of 5002 µg/plate.

For all strains and all conditions tested, the test item FC-131 showed negative results and can be considered as not mutagenic under the conditions of the test.