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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
purity; 89.7 % (w/w) (main component)

Method

Species / strain
Species / strain / cell type:
other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA
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 & experiment II
Vehicle / solvent:
- Vehicle/solvent used: DMSO
- 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
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation;


DURATION
- Preincubation period: 1 hour
- Exposure duration: 72 hours


NUMBER OF REPLICATIONS: Each concentration, including the controls, was tested in triplicate. 2 independent experiments were performed.


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:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Water solubility: not soluble
- Precipitation:
The test item precipitated in the overlay agar in the test tubes from 100 to 5000 µg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 333 to 5000 µg/plate. The undissolved particles had no influence on the data recording up to 2500 µg/plate. At 5000 µg/plate data recording was not possible, based on the precipitation. The test item precipitated in the overlay agar in the test tubes from 100 to 5000 µg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 333 to 5000 µg/plate. The undissolved particles had no influence on the data recording up to 2500 µg/plate. At 5000 µg/plate data recording was not possible, based on the precipitation.
- Other confounding effects: none
COMPARISON WITH HISTORICAL CONTROL DATA:
In experiment II, the data in the untreated control of strain TA 100 with and without S9 mix were slightly above our historical control range. Since this deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies and had no detrimental impact on the outcome of the study.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
Toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), were observed at the following concentrations (µg/plate):
Strain Experiment I Experiment II
without S9 mix with S9 mix without S9 mix with S9 mix
TA 1535 /* 333 – 2500 2500 2500
TA 1537 /* 2500 2500 /*
TA 98 /* 2500 2500 2500
TA 100 333 – 2500 333 – 2500 333 – 2500 1000 – 2500
WP2 uvrA /* /* /* /*
/ = no toxic effects; * at 5000 µg/plate analysis was not possible, based on the precipitation.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Summary Tabellen

Table1     Summary of Experiment I

Study Name: 1643301

Study Code: Harlan CCR 1643301

Experiment: 1643301 VV Plate

Date Plated: 28/07/2014

Assay Conditions:

Date Counted: 31/07/2014

 

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

DMSO

 

 

17 ± 1B M

9 ± 1

25 ± 6

181 ± 4

37 ± 5

Untreated

 

 

18 ± 3B M

9 ± 0

26 ± 7

181 ± 4

37 ± 8

test item

3 µg

 

17 ± 3B M

10 ± 3

23 ± 1

180 ± 0

35 ± 2

10 µg

 

19 ± 4B M

8 ± 2

24 ± 9

205 ± 34

35 ± 4

33 µg

 

20 ± 1B M

12 ± 3

30 ± 7

229 ± 8

34 ± 4

 

100 µg

 

15 ± 2B M

13 ± 1

23 ± 4

216 ± 25

39 ± 10

 

333 µg

 

11 ± 1B M P

13 ± 3P

34 ± 2P

69 ± 4P

32 ± 4P

 

1000 µg

 

10 ± 1B M P

7 ± 1P M

34 ± 3P M

18 ± 4P M

29 ± 6P M

 

2500 µg

 

9 ± 3B M P

5 ± 1P M

25 ± 6P M

7 ± 1P M

21 ± 3P M

 

5000 µg

 

 P N

 P N

 P N

 P N

 P N

NaN3

10 µg

 

2458 ± 52

 

 

1876 ± 45

 

4-NOPD

10 µg

 

 

 

319 ± 21

 

 

4-NOPD

50 µg

 

 

82 ± 9

 

 

 

MMS

2.0 µL

 

 

 

 

 

952 ± 41

 

 

 

 

 

 

 

 

 

With Activation

DMSO

 

 

14 ± 6

16 ± 5

35 ± 2

163 ± 9

51 ± 6

Untreated

 

 

12 ± 2

20 ± 1

44 ± 6

173 ± 6

48 ± 3

test item

3 µg

 

13 ± 3

20 ± 2

37 ± 8

173 ± 13

48 ± 7

10 µg

 

13 ± 4

15 ± 5

31 ± 1

182 ± 7

51 ± 10

33 µg

 

13 ± 2

17 ± 6

32 ± 10

191 ± 16

49 ± 7

 

100 µg

 

8 ± 2

14 ± 2

39 ± 11

151 ± 21

50 ± 10

 

333 µg

 

6 ± 1P

15 ± 2P

52 ± 10P M

47 ± 14P

39 ± 5P

 

1000 µg

 

6 ± 2P M

10 ± 1P M

28 ± 4P M

17 ± 3P M

25 ± 6P M

 

2500 µg

 

4 ± 1P M

5 ± 2P M

14 ± 2P M

5 ± 2P M

25 ± 2P M

 

5000 µg

 

 P N

 P N

 P N

 P N

 P N

2-AA

2.5 µg

 

321 ± 23

433 ± 10

3836 ± 249

3785 ± 124

 

2-AA

10.0 µg

 

 

 

 

 

195 ± 17

 

 

 

 

 

 

 

 

 

 

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

N

B

Precipitate

Manual count

Analysis not possible

Extensive bacterial growth

 

 


Table2     Summary of Experiment II

Study Name: 1643301

Study Code: Harlan CCR 1643301

Experiment: 1643301 HV2 Pre

Date Plated: 11/08/2014

Assay Conditions:

Date Counted: 14/08/2014

 

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

DMSO

 

 

21 ± 5B M

11 ± 1

24 ± 4

170 ± 10

38 ± 6

Untreated

 

 

23 ± 2B M

14 ± 6

28 ± 2

222 ± 37

47 ± 5

test item

3 µg

 

21 ± 1B M

12 ± 1

23 ± 1

171 ± 8

36 ± 4

10 µg

 

18 ± 2B M

7 ± 1

23 ± 3

165 ± 22

42 ± 7

33 µg

 

16 ± 1B M

9 ± 2

24 ± 5

187 ± 26

49 ± 3

 

100 µg

 

24 ± 2B M

6 ± 1

24 ± 4

121 ± 9

30 ± 1

 

333 µg

 

13 ± 2B M P

10 ± 2P

23 ± 5P

65 ± 8P

32 ± 2P

 

1000 µg

 

10 ± 1B M P

5 ± 3P

27 ± 2P M

51 ± 7P

19 ± 2P M

 

2500 µg

 

4 ± 1B M P

3 ± 1P M

3 ± 1P M

10 ± 2P M

19 ± 2P M

 

5000 µg

 

 B P N

 P N

 P N

 P N

 P N

NaN3

10 µg

 

2683 ± 144

 

 

1795 ± 276

 

4-NOPD

10 µg

 

 

 

358 ± 12

 

 

4-NOPD

50 µg

 

 

89 ± 9

 

 

 

MMS

2.0 µL

 

 

 

 

 

601 ± 60

 

 

 

 

 

 

 

 

 

With Activation

DMSO

 

 

9 ± 1

11 ± 2

25 ± 4

168 ± 10

44 ± 9

Untreated

 

 

15 ± 2

15 ± 3

28 ± 5

218 ± 33

58 ± 10

test item

3 µg

 

10 ± 3

11 ± 1

27 ± 7

169 ± 3

49 ± 13

10 µg

 

9 ± 3

12 ± 5

30 ± 1

149 ± 33

53 ± 4

33 µg

 

9 ± 4

13 ± 3

30 ± 8

152 ± 18

40 ± 5

 

100 µg

 

10 ± 3

14 ± 2

42 ± 8

109 ± 29

47 ± 14

 

333 µg

 

6 ± 1P

16 ± 1P

46 ± 4P M

85 ± 11P

40 ± 7P

 

1000 µg

 

8 ± 1P M

6 ± 1P M

16 ± 1P M

42 ± 3P M

29 ± 4P

 

2500 µg

 

4 ± 1P M

5 ± 1P M

10 ± 1P M

9 ± 2P M

26 ± 2P M

 

5000 µg

 

 P N

 P N

 P N

 P N

 P N

2-AA

2.5 µg

 

422 ± 42

171 ± 33

3670 ± 265

3817 ± 1049

 

2-AA

10.0 µg

 

 

 

 

 

205 ± 16

 

 

 

 

 

 

 

 

 

 

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

N

B

Precipitate

Manual count

Analysis not possible

Extensive bacterial growth

Applicant's summary and conclusion

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

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 metabolic activation.
Executive summary:

This study was performed to investigate the potential of the test item to induce gene muta­tions according to the plate incorporation test (experiment I) and the pre-incubation test (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 in both experiments:         3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The test item precipitated in the overlay agar in the test tubes from 100 to 5000 µg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 333 to 5000 µg/plate. The undissolved particles had no influence on the data recording up to 2500 µg/plate. At 5000 µg/plate data recording was not possible, based on the precipitation.

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.

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed in nearly all strains with and without metabol­ic activation.

No substantial increase in revertant colony numbers of any of the fivetester strains was observed following treatment with the test item 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.