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Toxicological information

Genetic toxicity: in vitro

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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:
21 February 2014 to 26 March 2014
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:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Guideline:
other: The Japanese Ministry of Economy Trade and Industry (METI), Ministry of Health, Labour and Welfare (MHLW) and Ministry of the Environment (MOE) Guidelines of 31 March 2011
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: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Identification: FAT 40863/A TE
Batch: BOP 02-13 (BS-ROE1670)
Appearance: Brownish powder, solid at 20°C
Purity: 82.1 %, dose calculation not adjusted to purity
Stability in solvent: Stable during this short time study in a range of pH 5 to 8
Retest date: 05 June 2018
Storage Conditions: At room temperature

Method

Target gene:
Histidine for Salmonella.
Tryptophan for E.Coli
Species / strain
Species / strain / cell type:
other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA
Additional strain / cell type 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
10; 33; 100; 333; 1000; 2500; and 5000 µg/plate / experiment II
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: suspended in deionised water
- Justification for choice of solvent/vehicle: best suitable solvent
Controls
Untreated negative controls:
yes
Negative solvent / vehicle 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 experimental 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
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS

- Water solubility: soluble to 50 mg/mL
- Precipitation:
No precipitation of the test item occurred up to the highest investigated dose.
- Other confounding effects: none
COMPARISON WITH HISTORICAL CONTROL DATA: performed no deviation
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 the test groups with and without metabolic activation.
Remarks on result:
other:
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

   Table1     Summary of Experiment I

Study Name: 1610201

Study Code: Harlan CCR 1610201

Experiment: 1610201 VV Plate

Date Plated: 21/02/2014

Assay Conditions:

Date Counted: 24/02/2014

 

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

 

 

21 ± 2

10 ± 2

26 ± 4

96 ± 5

53 ± 9

Untreated

 

 

18 ± 2

7 ± 2

25 ± 5

113 ± 9

50 ± 4

FAT 40863/A TE

3 µg

 

20 ± 7

13 ± 4

28 ± 5

114 ± 10

58 ± 9

 

10 µg

 

17 ± 2

9 ± 3

23 ± 3

116 ± 15

56 ± 11

 

33 µg

 

22 ± 2

11 ± 2

27 ± 8

100 ± 8

58 ± 8

 

100 µg

 

19 ± 3

11 ± 2

29 ± 6

116 ± 11

59 ± 11

 

333 µg

 

16 ± 1

10 ± 5

29 ± 5

106 ± 6

54 ± 0

 

1000 µg

 

16 ± 3D

10 ± 5D

23 ± 2D

103 ± 5D

63 ± 5D

 

2500 µg

 

14 ± 8D

10 ± 3D

26 ± 4D

109 ± 4D

60 ± 7D

 

5000 µg

 

15 ± 3D

7 ± 2D

22 ± 4D

92 ± 7D

51 ± 7D

NaN3

10 µg

 

3219 ± 204

 

 

2409 ± 58

 

4-NOPD

10 µg

 

 

 

311 ± 16

 

 

4-NOPD

50 µg

 

 

65 ± 6

 

 

 

MMS

2.0 µL

 

 

 

 

 

1268 ± 64

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

With Activation

Deionised water

 

 

15 ± 3

12 ± 3

43 ± 10

125 ± 14

63 ± 8

Untreated

 

 

17 ± 8

14 ± 2

50 ± 2

133 ± 14

69 ± 3

FAT 40863/A TE

3 µg

 

13 ± 2

12 ± 2

41 ± 12

130 ± 18

69 ± 3

 

10 µg

 

14 ± 2

11 ± 4

32 ± 4

129 ± 12

70 ± 5

 

33 µg

 

14 ± 3

14 ± 3

33 ± 3

123 ± 5

64 ± 5

 

100 µg

 

14 ± 2

16 ± 1

41 ± 6

130 ± 15

70 ± 5

 

333 µg

 

13 ± 3

15 ± 3

41 ± 7

147 ± 11

71 ± 12

 

1000 µg

 

9 ± 1D

9 ± 3D

38 ± 2D

126 ± 6D

74 ± 9D

 

2500 µg

 

10 ± 5D

15 ± 2D

27 ± 5D

127 ± 20D

69 ± 2D

 

5000 µg

 

13 ± 3D

13 ± 6D

29 ± 1D

136 ± 9D

75 ± 3D

2-AA

2.5 µg

 

597 ± 23

404 ± 47

3911 ± 185

4642 ± 84

 

2-AA

10.0 µg

 

 

 

 

 

321 ± 22

 

 

 

 

 

 

 

 

 

 

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

D

Densely coloured plate

 

 


Table2     Summary of Experiment II

Study Name: 1610201

Study Code: Harlan CCR 1610201

Experiment: 1610201 HV2 Pre

Date Plated: 20/03/2014

Assay Conditions:

Date Counted: 26/03/2014

 

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

 

 

18 ± 5

8 ± 1

22 ± 6

97 ± 11

59 ± 5

Untreated

 

 

14 ± 3

9 ± 3

30 ± 5

104 ± 11

52 ± 10

FAT 40863/A TE

10 µg

 

15 ± 4

11 ± 4

22 ± 7

105 ± 3

49 ± 1

 

33 µg

 

18 ± 8

10 ± 3

20 ± 4

90 ± 3

50 ± 2

 

100 µg

 

16 ± 1

10 ± 4

22 ± 3

97 ± 8

53 ± 9

 

333 µg

 

15 ± 2

9 ± 3

23 ± 5

93 ± 20

48 ± 2

 

1000 µg

 

14 ± 5D

9 ± 3D

23 ± 6D

99 ± 9D

63 ± 3D

 

2500 µg

 

16 ± 4D

7 ± 3D

18 ± 2D

94 ± 13D

55 ± 2D

 

5000 µg

 

16 ± 7D

5 ± 2D

22 ± 2D

92 ± 12D

43 ± 2D

NaN3

10 µg

 

2866 ± 197

 

 

1905 ± 137

 

4-NOPD

10 µg

 

 

 

271 ± 16

 

 

4-NOPD

50 µg

 

 

57 ± 12

 

 

 

MMS

2 µL

 

 

 

 

 

757 ± 36

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

With Activation

Deionised water

 

 

20 ± 8

26 ± 4

50 ± 6

103 ± 11

55 ± 0

Untreated

 

 

24 ± 9

25 ± 7

50 ± 2

105 ± 10

69 ± 9

FAT 40863/A TE

10 µg

 

18 ± 9

30 ± 15

50 ± 6

101 ± 3

58 ± 6

 

33 µg

 

18 ± 2

31 ± 6

51 ± 12

112 ± 9

66 ± 5

 

100 µg

 

20 ± 8

30 ± 3

45 ± 4

122 ± 13

61 ± 6

 

333 µg

 

21 ± 4

31 ± 12

44 ± 4

110 ± 11

70 ± 3

 

1000 µg

 

16 ± 3D

22 ± 7D

50 ± 7D

112 ± 13D

65 ± 4D

 

2500 µg

 

20 ± 3D

27 ± 5D

52 ± 5D

118 ± 6D

53 ± 3D

 

5000 µg

 

20 ± 4D

24 ± 3D

47 ± 3D

123 ± 4D

61 ± 11D

2-AA

2.5 µg

 

 

 

 

1426 ± 120

 

2-AA

2.5 µg

 

421 ± 83

194 ± 11

 

 

 

2-AA

10 µg

 

 

 

 

 

888 ± 81

Congo red

500 µg

 

 

 

350 ± 55

 

 

 

 

 

 

 

 

 

 

 

 

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

D

Densely coloured plate

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 with and without rat and hamster S9.
Executive summary:

This study was performed to investigate the potential of FAT 40863/A TE to induce gene muta­tions according to the plate incorporation assay with rat liver S9 (experiment I), and the pre-incubation test with hamster liver S9 (experiment II) using theSalmonella typhimuriumstrains TA 1535, TA 1537, TA 98, TA 100, and theEscherichia 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:                                10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

No precipitation of the test item occurred up to the highest investigated dose.

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 the test groups with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with FAT 40863/A TE at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct in­crease of induced revertant colonies.