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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:
2010 -08-09 till 2010-08-24
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:
2010
Report date:
2010

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Automatically generated during migration to IUCLID 6, no data available
IUPAC Name:
Automatically generated during migration to IUCLID 6, no data available
Details on test material:
Identity: Fluowet El 812
CAS No. 85995-91-1
Batch No.: T 7362
Description: wax like solid
Colour: white to slight violet
Purity: 92.5 %
Dose calculations adjusted to purity
Stability in solvent: Not indicated by the sponsor
Storage: At room temperature, light protected
Expiration Date: October 28, 2011

Method

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
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: Acetone
- Justification for choice of solvent/vehicle: recommended by the sponsor
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
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate incorporation; preincubation;


DURATION
- Preincubation period: 1 hour
- 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
- Precipitation:
The test item precipitated in the overlay agar in the test tubes from 1000 µg/plate up to 5000 µg/plate in experiment I and from 2500 µg/plate up to 5000 µg/plate in experiment II. Precipitation on the incubated agar plates was observed from 1000 µg/plate up to 5000 µg/plate in both experiments with and without S9 mix. The undissolved particles had no influence on the data recording.
- Other confounding effects:
COMPARISON WITH HISTORICAL CONTROL DATA: performed
ADDITIONAL INFORMATION ON CYTOTOXICITY: no toxic effects observed
Remarks on result:
other: other: reverse mutation assay
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

1.1     Summary of Results Pre-Experiment and Experiment I

Study Name: 1358102

Study Code: Harlan CCR 1358102

Experiment: 1358102 VV Plate

Date Plated: 09/08/2010

Assay Conditions:

Date Counted: 12/08/2010 to 16/08/2010

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

Acetone

13 ± 1

7 ± 1

27 ± 5

147 ± 6

55 ± 4

Untreated

18 ± 3

10 ± 2

29 ± 5

133 ± 10

61 ± 11

Fluowet El 812

3 µg

12 ± 5

8 ± 5

24 ± 5

138 ± 19

55 ± 1

10 µg

16 ± 3

8 ± 2

29 ± 2

152 ± 8

63 ± 2

33 µg

14 ± 5

8 ± 1

28 ± 1

137 ± 13

57 ± 6

100 µg

13 ± 4

10 ± 3

30 ± 4

136 ± 4

56 ± 6

333 µg

14 ± 2

9 ± 1

28 ± 4

137 ± 4

63 ± 10

1000 µg

13 ± 3P

7 ± 3P

32 ± 3P

136 ± 11P

54 ± 3P

2500 µg

13 ± 2P M

5 ± 2P M

24 ± 1P M

149 ± 17P

60 ± 8P

5000 µg

11 ± 2P M

5 ± 2P M

15 ± 2P M

128 ± 8P M

42 ± 1P M

NaN3

10 µg

1721 ± 110

1935 ± 118

4-NOPD

10 µg

301 ± 17

4-NOPD

50 µg

67 ± 4

MMS

3.0 µL

1170 ± 22

With Activation

Acetone

23 ± 3

14 ± 2

36 ± 9

164 ± 14

66 ± 10

Untreated

20 ± 6

17 ± 2

39 ± 11

151 ± 23

78 ± 11

Fluowet El 812

3 µg

21 ± 8

12 ± 6

40 ± 9

157 ± 6

70 ± 8

10 µg

24 ± 3

12 ± 2

38 ± 3

141 ± 5

81 ± 8

33 µg

19 ± 6

13 ± 2

40 ± 12

141 ± 12

73 ± 8

100 µg

19 ± 4

13 ± 2

37 ± 7

151 ± 4

80 ± 1

333 µg

26 ± 6

17 ± 2

48 ± 4

140 ± 9

81 ± 9

1000 µg

24 ± 9P

16 ± 1P

35 ± 6P M

158 ± 4P

86 ± 9P

2500 µg

20 ± 4P M

13 ± 3P M

35 ± 6P M

170 ± 3P

70 ± 7P M

5000 µg

13 ± 3P M

7 ± 1P M

25 ± 7P M

131 ± 16P M

69 ± 9P M

2-AA

2.5 µg

358 ± 5

545 ± 41

2089 ± 430

3173 ± 59

2-AA

10.0 µg

305 ± 48

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

P

M

Precipitate

Manual count


1.2     Summary of Results Experiment II

Study Name: 1358102

Study Code: Harlan CCR 1358102

Experiment: 1358102 HV2 Pre

Date Plated: 18/08/2010

Assay Conditions:

Date Counted: 24/08/2010

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

Acetone

13 ± 6

18 ± 5

32 ± 10

117 ± 16

45 ± 3

Untreated

14 ± 2

16 ± 3

35 ± 3

130 ± 16

43 ± 9

Fluowet El 812

10 µg

12 ± 3

16 ± 5

34 ± 3

129 ± 1

47 ± 1

33 µg

13 ± 2

15 ± 2

32 ± 9

124 ± 8

43 ± 3

100 µg

13 ± 2

16 ± 1

29 ± 4

120 ± 20

48 ± 1

333 µg

12 ± 4

11 ± 4

35 ± 5

129 ± 19

39 ± 10

1000 µg

17 ± 2P

18 ± 3P

37 ± 3P

131 ± 7P

41 ± 5P

2500 µg

9 ± 3P M

16 ± 1P

33 ± 3P M

151 ± 5P

37 ± 3P M

5000 µg

9 ± 3P M

12 ± 2P M

24 ± 5P M

133 ± 7P M

36 ± 3P M

NaN3

10 µg

1767 ± 37

2020 ± 98

4-NOPD

10 µg

433 ± 26

4-NOPD

50 µg

78 ± 5

MMS

3.0 µL

498 ± 21

With Activation

Acetone

24 ± 7

20 ± 3

47 ± 7

133 ± 9

58 ± 4

Untreated

26 ± 1

29 ± 6

43 ± 8

121 ± 5

56 ± 11

Fluowet El 812

10 µg

26 ± 7

25 ± 4

47 ± 8

138 ± 7

54 ± 16

33 µg

19 ± 5

24 ± 3

47 ± 2

141 ± 13

64 ± 8

100 µg

24 ± 6

26 ± 3

40 ± 3

139 ± 33

56 ± 6

333 µg

20 ± 4

25 ± 6

49 ± 9

118 ± 9

48 ± 7

1000 µg

23 ± 2P M

28 ± 2P

55 ± 5P

135 ± 16P

53 ± 12P

2500 µg

16 ± 4P M

17 ± 3P M

36 ± 1P M

133 ± 10P M

63 ± 5P

5000 µg

15 ± 3P M

13 ± 4P M

35 ± 5P M

122 ± 8P M

46 ± 5P M

2-AA

2.5 µg

350 ± 11

271 ± 11

1799 ± 45

1809 ± 39

2-AA

10.0 µg

395 ± 10

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

P

M

Precipitate

Manual count

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with and without S9 mix

In conclusion, it can be stated that during the described mutage¬nicity 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.
Executive summary:

The test item Fluowet El 812 was assessed for its potential to induce gene mutations in the plate incorporation test (experi­ment I) and the pre-incubation test (experiment II) using Salmo­nella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escheri­chia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were 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

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in both experiments.

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 Fluowet El 812 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. They showed a distinct in­crease of induced revertant colonies.