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:
2008-05-27 to 2008-06-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
, 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
, 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2007-01-19
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Naringin DHC; 1-[4-[[2-O-(6-Deoxy-L-mannopyranosyl)-D-glucopyranosyl]oxy]-2,6-dihdroxyphenyl]-3(4-hydroxyphenyl)-1-propanone
- Substance type: technical product
- Physical state: solid, whitish powder
- Analytical purity: 99 % [a/a]
- water content: 6.4 %
- Lot/batch No.: 2
- Expiration date of the lot/batch: January 2010
- Storage condition of test material: 15-25 °C, dark, don't freeze, keep away from strong light and heat

Method

Target gene:
the S. typhimurium histidine (his) system

- TA 1537: his C 3076; rfa-; uvrB-
- TA 98: his D 3052, rfa-, uvrB-; R.factor
- TA 1535: his G46; rfa-; uvrB-
- TA 102: his G 428; rfa-, uvrB+; R-factor
- TA 100: his G 46; rfa-, uvrB-, R-factor
Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 preparation with 31.4 mg/mL in the pre-experiment/experiment I , and 28.7 mg/mL in experiment II.
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 preparation with 31.4 mg/mL in the pre-experiment/experiment I , and 28.7 mg/mL in experiment II.
Test concentrations with justification for top dose:
- experiment I: 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate
- experiment II: 33, 100, 333, 1000, 2500, and 5000 µg/plate
Vehicle:
- Vehicle(s)/solvent(s) used: on the day of the experiment, the test item was dissolved in DMSO (purity < 99%)
- Justification for choice of solvent: the solvent was choosen because of its solubility and its relative nontoxicity to the bacteria
- precipitation was only observed in the test tubes at 5000 µg/plate in experiment I
- no further significant details stated
Controls
Negative controls:
yes
Remarks:
concurrent untreated and solvent controls
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: without metabolic activation: sodium azide (TA 1535, TA 100); 4-nitro-o-phenylene-diamine (TA1537, TA 98); Methyl methane sulfonate (TA 102). With metabolic activation: 2-aminoanthracene (TA 1535, TA 1537, TA 98, TA 100, TA 102)
Details on test system and conditions:
METHOD OF APPLICATION: in agar/plate incorporation (experiment I) and in agar/ pre incubation (experiment II)

MATERIAL MIX
- 100 µL test solution at each dose level (solvent or reference mutagen solution (positive control))
- 500 µL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation)
- 100 µL Bacteria suspension (cf. test system, pre-culture of the strains)
- 2000 µL overlay agar

METABOLIC ACTIVATION:
S9-preparation
Phenobarbital/ß-Naphthoflavone induced rat liver S9 is used as the metabolic activation system. The S9 is prepared from 8-12 weeks old male Wistar Hanlbm rats, weight approx. 220-320 g induced by applications of 80mg/kg body weight Phenobarbital i.p. and ß-Naphthoflavone p.o. each on three consecutive days. The livers are prepared 24 hours after the last treatment. The S9 fractions are produced by dilution of the liver homogenate with a KCl solution (1+3) followed by centrifugation at 9000 g. Aliquots of the supernatant are frozen and stored in ampoules at -80° C. Small numbers of the ampoules can be kept at -20°C for up to one week. Each batch of S9 mix is routinely tested with 2-aminoanthracene as well as benzo(a)pyrene.

S9-mix
Before the experiment an appropiate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 was 10% v/v in the S9 mix. Cofactors were added to the S9 mix to reach the following concentrations in the S9 mix: 8mM MgCl2, 33mM KCl, 5mM Glucose-6-phosphate and 5mM NADP in 100mM sodium-ortho-phosphate-buffer, pH 7.4.
DURATION
- Preincubation period: 60 minutes
- Exposure duration: at least 48 hours

DATA RECORDING:
The colonies were counted using the Petri Viewer Mk2 (Perceptive Instruments Ltd, Suffolk CB 7BN, UK) with the software program Ames Study Manager. The counter was connected to an IBM AT compatible PC with printer which printed out both, the individual and mean values of the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates (see tables of results). Due to reduced background growth, the colonies were partly counted manually.

NUMBER OF REPLICATIONS: three plates

- no further significant details stated
Evaluation criteria:
The Salmonella typhimurium reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of our historical data
- the positive control substances should produce a significant increase in mutant colony frequencies

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.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
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'.
Species / strain:
S. typhimurium TA 102
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:
- no significant details stated

Any other information on results incl. tables

- 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 and no toxic effects occurred
- no substantial increase in revertant colony numbers of any of the five strains was observed following treatment with
Naringin DHC at any concentration level with and without S9

Table 1: Summary of results pre-experiment and experiment I

Test group

Dose level

[µg/plate]

Revertant colony counts (mean ± SD)

TA 1535

TA 1537

TA 98

TA 100

TA 102

Without activation

DMSO

-

17±4

16±3

29±7

104±15

399±23

Untreated

-

9±3

11±2

31±4

124±9

390±31

Naringin DHC

3

14±7

13±1

25±3

118±16

390±5

10

12±5

18±4

23±1

121±8

454±26

33

14±4

17±3

36±4

124±9

451±31

100

15±2

13±3

31±10

122±10

445±11

333

12±4

13±3

30±5

123±7

414±37

1000

13±4

16±3

25±7

124±2

415±15

2500

11±5

11±3

21±2

114±5

436±26

5000

13±4

11±2

29±3

114±9

429±4

NaN3

10

1571±51

-

-

1729±85

-

4-NOPD

10

-

-

457±44

-

-

4-NOPD

50

-

113±15

-

-

-

MMS

3.0 µL

-

-

-

-

3619±123

With activation

DMSO

-

18±6

16±7

35±6

136±9

481±28

Untreated

-

16±5

17±4

37±4

134±8

510±13

Naringin DHC

3

21±2

16±7

43±7

133±6

480±9

10

20±12

18±1

42±6

147±3

492±32

33

21±5

16±4

37±8

143±11

519±20

100

21±1

17±6

38±4

141±17

512±25

333

14±2

14±2

33±8

144±13

488±19

1000

16±3

15±2

35±5

137±7

511±13

2500

15±8

18±5

30±2

153±8

528±15

5000

13±3

21±2

38±5

123±5

514±3

2-AA

2.5

264±30

285±50

1445±63

2247±48

-

2-AA

10.0

-

-

-

-

1958±47

Table 2: Summary of results experiment II

Test group

Dose level

[µg/plate]

Revertant colony counts (mean ± SD)

TA 1535

TA 1537

TA 98

TA 100

TA 102

Without activation

DMSO

-

13±1

10±2

27±4

116±7

476±13

Untreated

-

16±3

18±3

32±7

131±15

446±31

Naringin DHC

33

13±3

13±1

29±1

116±7

455±26

100

16±3

15±1

29±5

118±6

473±21

333

13±5

14±1

31±10

110±5

482±2

1000

17±4

12±3

31±3

115±4

458±15

2500

14±5

13±1

33±10

121±8

465±13

5000

12±2

12±2

34±4

111±9

461±8

NaN3

10

1775±35

-

-

2171±71

-

4-NOPD

10

-

-

466±17

-

-

4-NOPD

50

-

102±13

-

-

-

MMS

3.0 µL

-

-

-

-

1917±58

With activation

DMSO

-

18±5

16±5

33±11

137±5

560±30

Untreated

-

19±6

19±3

37±4

146±4

558±4

Naringin DHC

33

16±5

15±6

39±9

123±5

539±29

100

15±6

17±3

34±12

123±5

557±11

333

15±1

17±5

41±6

123±16

575±20

1000

16±2

15±2

37±8

116±13

551±36

2500

14±3

10±3

38±7

136±13

589±21

5000

19±5

10±3

43±2

121±12

600±38

2-AA

2.5

177±5

143±12

1107±107

1119±50

-

2-AA

10.0

-

-

-

-

2525±109

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the test conditions the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Either with and without metabolic activation.