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Diss Factsheets

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
end on 05-JUL-2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: study performed according to OECD guideline and GLP

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
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Potassium (R)-(4-hydroxyphenyl)[(3-methoxy-1-methyl-3-oxoprop-1-enyl)amino]acetate
EC Number:
273-992-8
EC Name:
Potassium (R)-(4-hydroxyphenyl)[(3-methoxy-1-methyl-3-oxoprop-1-enyl)amino]acetate
Cas Number:
69416-61-1
IUPAC Name:
potassium (4-hydroxyphenyl)[(3-methoxy-1-methyl-3-oxoprop-1-en-1-yl)amino]acetate
Details on test material:
- Name of test material (as cited in study report): D(-)-P-HydroxyphenylglycineDaneSalt (K,Methyl), Code Name: HPG-DS
- Substance type: monoconstituent substance
- Physical state: data not available
- Stability under test conditions: not indicated
- Storage condition of test material: at room temperature, light and moisture protected

Method

Species / strain
Species / strain / cell type:
other: Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/β-Naphthoflavone induced rat liver S9 is used as the metabolic activation system
Test concentrations with justification for top dose:
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
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: the solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9 mix: sodium azide (NaN3, 10µg/p) in TA1535 & TA100, 4-nitro-o-phenylene-diamine (4-NOPD, 10-50µg/p) in TA1537 & TA98, methyl methane sulfonate (MMS, 3µg/p) in TA 102 ; +S9 mix: 2-aminoanthracene (2-AA, 2.5-10µg/p)
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate incorporation test (experiment I) and the pre-incubation test (experiment II)

DURATION
- Preincubation period: 60 minutes (experiment II)
- Exposure duration: at least 48 hours at 37 °C in the dark

NUMBER OF REPLICATES: For each strain and dose level, including the controls three plates were used

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of revertants
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.

Results and discussion

Test results
Species / strain:
other: Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no precipitation of the test item occurred up to the highest investigated dose
- Effects of pH, Effects of osmolality, Evaporation from medium, Water solubility:


RANGE-FINDING/SCREENING STUDIES:
The pre-experiment is reported as main experiment I, since the following criteria are met: Evaluable plates (>0 colonies) at five concentrations or more in all strains used.

COMPARISON WITH HISTORICAL CONTROL DATA:
In experiment I and II with metabolic activation, the data in the solvent control of strain TA 102 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.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Summary of Results Pre-Experiment and Experiment I

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

Deionised water

 

 

15 ± 7

11 ± 1

31 ± 3

166 ± 20

472 ± 18

Untreated

 

 

16 ± 4

7 ± 1

31 ± 6

166 ± 9

492 ± 22

HPG - DS

3 µg

 

15 ± 8

11 ± 3

27 ± 3

160 ± 5

447 ± 46

 

10 µg

 

15 ± 4

15 ± 2

25 ± 4

154 ± 5

460 ± 12

 

33 µg

 

13 ± 4

14 ± 3

32 ± 7

158 ± 10

492 ± 42

 

100 µg

 

14 ± 4

13 ± 3

26 ± 5

196 ± 21

450 ± 14

 

333 µg

 

15 ± 3

10 ± 4

29 ± 5

171 ± 21

431 ± 11

 

1000 µg

 

16 ± 3

11 ± 2

33 ± 6

162 ± 7

463 ± 9

 

2500 µg

 

12 ± 3

8 ± 2

29 ± 5

170 ± 12

450 ± 34

 

5000 µg

 

14 ± 1

12 ± 2

28 ± 1

163 ± 15

357 ± 114

NaN3

10 µg

 

2059 ± 98

 

 

2165 ± 86

 

4-NOPD

10 µg

 

 

 

294 ± 4

 

 

4-NOPD

50 µg

 

 

81 ± 2

 

 

 

MMS

3.0 µL

 

 

 

 

 

3498 ± 218

 

 

 

 

 

 

 

 

 

With Activation

Deionised water

 

 

20 ± 4

16 ± 5

37 ± 3

174 ± 14

695 ± 24

Untreated

 

 

18 ± 7

21 ± 4

36 ± 7

199 ± 23

644 ± 11

HPG - DS

3 µg

 

16 ± 1

15 ± 7

35 ± 6

183 ± 11

678 ± 30

 

10 µg

 

15 ± 5

16 ± 2

40 ± 6

184 ± 13

687 ± 21

 

33 µg

 

19 ± 5

9 ± 5

41 ± 3

189 ± 9

682 ± 31

 

100 µg

 

18 ± 4

10 ± 2

38 ± 7

196 ± 21

607 ± 65

 

333 µg

 

23 ± 7

18 ± 3

35 ± 8

190 ± 11

602 ± 32

 

1000 µg

 

17 ± 3

18 ± 3

46 ± 7

191 ± 19

650 ± 47

 

2500 µg

 

19 ± 11

11 ± 3

36 ± 4

206 ± 3

649 ± 6

 

5000 µg

 

22 ± 5

13 ± 1

36 ± 9

203 ± 6

646 ± 34

2-AA

2.5 µg

 

497 ± 57

669 ± 44

3679 ± 223

4964 ± 175

 

2-AA

10.0 µg

 

 

 

 

 

2736 ± 318

Table 2: Summary of Results Experiment II

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

Deionised water

 

 

11 ± 3

10 ± 4

35 ± 2

175 ± 16

432 ± 17

Untreated

 

 

13 ± 0

15 ± 4

38 ± 4

150 ± 8

414 ± 16

HPG - DS

33 µg

 

12 ± 3

14 ± 1

33 ± 6

169 ± 11

365 ± 42

 

100 µg

 

11 ± 4

7 ± 1

33 ± 2

167 ± 7

415 ± 48

 

333 µg

 

11 ± 3

12 ± 2

32 ± 3

168 ± 19

401 ± 36

 

1000 µg

 

13 ± 1

13 ± 4

38 ± 12

157 ± 11

437 ± 40

 

2500 µg

 

12 ± 2

9 ± 3

32 ± 6

188 ± 40

439 ± 18

 

5000 µg

 

9 ± 3

12 ± 6

34 ± 2

169 ± 6

447 ± 34

NaN3

10 µg

 

1857 ± 126

 

 

2063 ± 192

 

4-NOPD

10 µg

 

 

 

392 ± 8

 

 

4-NOPD

50 µg

 

 

85 ± 13

 

 

 

MMS

3.0 µL

 

 

 

 

 

2160 ± 628

 

 

 

 

 

 

 

 

 

With Activation

Deionised water

 

 

25 ± 4

14 ± 2

44 ± 8

199 ± 18

719 ± 50

Untreated

 

 

26 ± 7

15 ± 2

57 ± 6

192 ± 13

624 ± 40

HPG - DS

33 µg

 

27 ± 7

14 ± 1

48 ± 8

186 ± 29

660 ± 37

 

100 µg

 

21 ± 7

12 ± 3

43 ± 5

216 ± 14

680 ± 64

 

333 µg

 

26 ± 3

15 ± 2

49 ± 1

211 ± 24

603 ± 73

 

1000 µg

 

24 ± 4

15 ± 1

53 ± 2

204 ± 3

650 ± 25

 

2500 µg

 

20 ± 6

14 ± 2

48 ± 4

195 ± 30

636 ± 43

 

5000 µg

 

22 ± 13

8 ± 2

47 ± 11

205 ± 19

501 ± 116

2-AA

2.5 µg

 

454 ± 31

541 ± 36

3619 ± 270

3505 ± 257

 

2-AA

10.0 µg

 

 

 

 

 

2420 ± 133

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

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.
Executive summary:

In a reverse gene mutation assay in bacteria (OECD 471, GLP), strains TA 1535, TA 1537, TA 98, TA 100, and TA 102 of S. typhimurium were exposed to D(-)-P-HydroxyphenylglycineDaneSalt (K,Methyl), Code Name: HPG-DS in deionised water in the presence and absence of mammalian metabolic activation at the following concentrations:

- Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate (plate incorporation)

- Experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate (pre-incubation)

D(-)-P-HydroxyphenylglycineDaneSalt (K,Methyl), Code Name: HPG-DS was tested up to limit concentration (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 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 D(-)-P-HydroxyphenylglycineDaneSalt (K,Methyl), Code Name: HPG-DS at any concentration 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 in-crease in induced revertant colonies.

 

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.

 

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.