Genetic toxicity in vitro

Jump to

Administrative data

Confidentiality
[Not publishable]
Justification
[Not publishable]
Use restricted to selected regulatory programmes
[Not publishable]
Endpoint
in vitro gene mutation study in bacteria

Type of genotoxicity: gene mutation

Type of information
experimental study
Adequacy of study
key study
Robust study summary
[Not publishable]
Used for classification
[Not publishable]
Used for SDS
[Not publishable]
Study period: start date
04-Nov-2013
End date
03-Dec-2013
Remark

2013 -11-04 till 2013-12-03

Reliability
1 (reliable without restriction)
Rationale for reliability incl. deficiencies
other: Guideline-conform study under GLP without deviations
Data waiving
[Empty]
Justification for data waiving
[Empty]
Justification for type of information
[Empty]

Attached justification

Cross-reference

Data source

Reference
Data access
[Not publishable]
Data protection claimed
[Not publishable]

Materials and methods

Test guideline

#1 - Test guideline

Qualifier
according to guideline
Guideline
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks

[Empty]

Deviations
no
Principles of method if other than guideline
[Empty]
GLP compliance
yes (incl. QA statement)
Type of assay
bacterial reverse mutation assay

Test material

Test material information
Test material information
Additional test material information
[Empty]
Specific details on test material used for the study
[Empty]
Specific details on test material used for the study (confidential)
[Not publishable]

Method

Target gene

[Empty]

Species / strain

#1 - Species / strain

Species / strain / cell type
other: TA 1535, TA 1537, TA 98, TA 100, TA 102
Details on mammalian cell type (if applicable)
[Empty]
Additional strain / cell type characteristics
not applicable
Cytokinesis block (if used)

[Empty]

Metabolic activation
with and without
Metabolic activation system

Phenobarbital/ß-Naphthoflavone induced rat liver S9

Test concentrations

[Empty]

High dose level used
[Empty]
Justification for deviation from the high dose level
[Not publishable]
Vehicle / solvent

- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: soluble in DMSO

Controls

#1 - 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
Remarks

[Empty]

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.

Rationale for test conditions

[Empty]

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 TA 102 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.

Model and software

Model name and version

[Empty]

Software name and version

[Empty]

Remarks
[Not publishable]

Any other information on materials and methods incl. tables

[Not publishable]

Results and discussion

Test results

#1 - Test results

Key result
Species / strain
other: TA 1535, TA 1537, TA 98, TA 100, TA 102
Metabolic activation
with and without
Genotoxicity
negative
Cytotoxicity / choice of top concentrations
no cytotoxicity
Vehicle controls validity
valid
Untreated negative controls validity
valid
True negative controls validity
[Empty]
Positive controls validity
valid
Additional information on results

TEST-SPECIFIC CONFOUNDING FACTORS

- Water solubility: unsoluble in water

- Precipitation: No precipitation observed

- Other confounding effects:

COMPARISON WITH HISTORICAL CONTROL DATA: performed

In experiment II, the number of colonies did not quite reach the lower limit of our historical control data in strain TA 100 in the solvent control with and without metabolic activation. Since this deviation is rather small, this effect is judged to be based upon statistical fluctuations and has no detrimental impact on the outcome of the study.

ADDITIONAL INFORMATION ON CYTOTOXICITY:

The plates incubated with the test item showed normal back¬ground 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 metabol¬ic activation.

Overall remarks on result
other: reverse mutation assay

Additional information about applicability domain and reliability of (Q)SAR predictions

Fit with the applicability domain
[Empty]
Justification for the fit with the applicability domain

[Empty]

Fit with the space defined by the training set of the model
[Empty]
Mechanistic and metabolic considerations

[Empty]

Similar substances with experimental data

Performance of the model for similar substances

[Empty]

Conclusions on applicability domain and reliability
[Empty]
Uncertainty
[Empty]

Any other information on results incl. tables

Table1     Summary of Experiment I

Study Name: 1584504

Study Code: Harlan CCR 1584504

Experiment: 1584504 VV Plate

Date Plated: 04/11/2013

Assay Conditions:

Date Counted: 07/11/2013

 

Metabolic

Activation

Test

Group

Dose Level

(per plate)

 

Revertant Colony Counts (Mean ±SD)

 

 

 

 

 

 

 

 

 

 

 

 

 

TA 1535

TA 1537

TA 98

TA 100

TA 102

 

 

 

 

 

 

 

 

 

Without Activation

DMSO

 

 

20 ± 4

12 ± 2

24 ± 3

97 ± 8

498 ± 13

Untreated

 

 

22 ± 2

10 ± 5

24 ± 5

102 ± 8

477 ± 1

(-)-Shikimic acid

3 µg

 

18 ± 6

12 ± 3

23 ± 2

99 ± 11

541 ± 18

 

10 µg

 

19 ± 4

12 ± 2

26 ± 6

97 ± 14

483 ± 17

 

33 µg

 

20 ± 3

11 ± 1

24 ± 4

104 ± 10

526 ± 17

 

100 µg

 

22 ± 3

8 ± 2

24 ± 4

88 ± 9

529 ± 16

 

333 µg

 

24 ± 0

12 ± 5

24 ± 4

100 ± 22

420 ± 90

 

1000 µg

 

24 ± 8

10 ± 3

26 ± 4

98 ± 4

498 ± 30

 

2500 µg

 

20 ± 5

12 ± 2

24 ± 2

104 ± 10

497 ± 2

 

5000 µg

 

13 ± 4

15 ± 4

30 ± 3

103 ± 3

476 ± 11

NaN3

10 µg

 

2921 ± 77

 

 

2207 ± 29

 

4-NOPD

10 µg

 

 

 

269 ± 46

 

 

4-NOPD

50 µg

 

 

72 ± 12

 

 

 

MMS

2.0 µL

 

 

 

 

 

5384 ± 568

 

 

 

 

 

 

 

 

 

With Activation

DMSO

 

 

28 ± 5

16 ± 4

34 ± 9

127 ± 13

590 ± 25

Untreated

 

 

32 ± 1

21 ± 10

39 ± 4

141 ± 7

594 ± 14

(-)-Shikimic acid

3 µg

 

28 ± 8

18 ± 1

29 ± 8

129 ± 8

605 ± 4

 

10 µg

 

34 ± 7

14 ± 1

34 ± 1

133 ± 28

581 ± 45

 

33 µg

 

25 ± 10

17 ± 3

37 ± 1

142 ± 12

578 ± 17

 

100 µg

 

32 ± 8

18 ± 2

32 ± 4

127 ± 3

629 ± 29

 

333 µg

 

29 ± 1

19 ± 4

38 ± 3

124 ± 3

544 ± 33

 

1000 µg

 

31 ± 4

21 ± 4

41 ± 6

120 ± 8

531 ± 35

 

2500 µg

 

24 ± 2

16 ± 6

38 ± 4

119 ± 4

640 ± 98

 

5000 µg

 

33 ± 6

14 ± 6

33 ± 0

112 ± 6

605 ± 15

2-AA

2.5 µg

 

536 ± 40

340 ± 21

4086 ± 165

4271 ± 168

 

2-AA

10.0 µg

 

 

 

 

 

5347 ± 502

 

 

 

 

 

 

 

 

 

 

Key to Positive Controls

 

 

 

NaN3

2-AA

MMS

4-NOPD

sodium azide

2-aminoanthracene

methyl methane sulfonate

4-nitro-o-phenylene-diamine

 

 

 

 


Table2     Summary of Experiment II

Study Name: 1584504

Study Code: Harlan CCR 1584504

Experiment: 1584504 HV2 Pre

Date Plated: 28/11/2013

Assay Conditions:

Date Counted: 03/12/2013

 

Metabolic

Activation

Test

Group

Dose Level

(per plate)

 

Revertant Colony Counts (Mean ±SD)

 

 

 

 

 

 

 

 

 

 

 

 

 

TA 1535

TA 1537

TA 98

TA 100

TA 102

 

 

 

 

 

 

 

 

 

Without Activation

DMSO

 

 

21 ± 6

9 ± 3

23 ± 11

75 ± 4

381 ± 12

Untreated

 

 

25 ± 10

9 ± 5

24 ± 6

94 ± 10

479 ± 39

(-)-Shikimic acid

33 µg

 

24 ± 2

7 ± 2

20 ± 3

82 ± 12

425 ± 52

 

100 µg

 

24 ± 8

9 ± 4

21 ± 2

75 ± 8

376 ± 19

 

333 µg

 

28 ± 3

7 ± 3

21 ± 4

88 ± 10

398 ± 20

 

1000 µg

 

24 ± 4

7 ± 2

25 ± 8

85 ± 7

376 ± 31

 

2500 µg

 

21 ± 2

10 ± 4

24 ± 5

71 ± 4

334 ± 43

 

5000 µg

 

22 ± 8

8 ± 5

26 ± 2

85 ± 13

381 ± 54

NaN3

10 µg

 

2744 ± 64

 

 

2045 ± 197

 

4-NOPD

10 µg

 

 

 

411 ± 9

 

 

4-NOPD

50 µg

 

 

83 ± 2

 

 

 

MMS

2.0 µL

 

 

 

 

 

3599 ± 221

 

 

 

 

 

 

 

 

 

With Activation

DMSO

 

 

17 ± 4

22 ± 8

41 ± 8

84 ± 11

438 ± 15

Untreated

 

 

18 ± 4

18 ± 4

32 ± 9

94 ± 3

418 ± 11

(-)-Shikimic acid

33 µg

 

19 ± 8

17 ± 5

38 ± 9

88 ± 8

395 ± 9

 

100 µg

 

18 ± 8

20 ± 5

33 ± 9

75 ± 5

441 ± 29

 

333 µg

 

18 ± 4

14 ± 6

41 ± 6

94 ± 17

411 ± 74

 

1000 µg

 

14 ± 5

17 ± 4

29 ± 6

92 ± 6

470 ± 15

 

2500 µg

 

22 ± 4

20 ± 9

30 ± 3

88 ± 10

451 ± 62

 

5000 µg

 

19 ± 4

17 ± 6

41 ± 3

96 ± 13

388 ± 20

2-AA

2.5 µg

 

510 ± 23

224 ± 18

2401 ± 292

1969 ± 90

 

2-AA

10.0 µg

 

 

 

 

 

986 ± 70

 

 

 

 

 

 

 

 

 

 

Key to Positive Controls

 

 

 

NaN3

2-AA

MMS

4-NOPD

sodium azide

2-aminoanthracene

methyl methane sulfonate

4-nitro-o-phenylene-diamine

 

 

 

Overall remarks, attachments

Overall remarks
[Not publishable]

Attachments

Applicant's summary and conclusion

Conclusions

Interpretation of results: negative with and without metabolic activation

In conclusion, it can be stated that during the described mutage¬nicity test and under the experimental conditions reported, the (-)-Shikimic acid did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Executive summary

This study was performed to investigate the potential of (-)-Shikimic acid 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 TA 102.

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:                                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 fivetester strains was observed following treatment with (-)-Shikimic acid 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.