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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 study, GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material: DOPA-Glycinate

Method

Target gene:
his (Salmonella strains), trp (E. coli strain)
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
other: Amino-acid requiring strains, histidine for S. typhimurium and tryptophane for E. coli
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
First assay: 0, 62, 185, 556, 1667 and 5000 µg/plate (in the presence and in the absence of rat liver S9 fraction)
Second assay: 0, 12.5, 25, 50, 100 and 200 µg/plate (in the presence and in the absence of rat liver S9 fraction)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Milli-Q-water
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Remarks:
Solvent (Milli-Q-water)
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
ethylnitrosurea
other: (Absence of S9-mix)
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 2-aminoanthracene; (Presence of S9-mix)
Details on test system and experimental conditions:
Way of application:
Test substance was dissolved in Milli-Q-water at 50 mg/mL. The stock solution was sterilized by passage through a micropore filter (0.45 µM).
For the plate incorporation tests, 0.1 mL of a fully grown culture of the appropriate strain, 0.1 mL of the appropriate test substance solution and 0.5 mL S9-mix for the tests with metabolic activation or 0.5 mL sodium phosphate for the tests without metabolic activation were added to top agar mix and poured onto minimal glucose agar plates. All determinations were made in triplicate. The cultures were incubated at 37°C for 48–72 hours.

Pre-incubation time:
None

Other modifications:
None

Examinations:
Number of revertant colonies

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Genotoxicity
Without metabolic activation: test item dissolved in Milli-Q-water did not cause a two-fold or more increase in the mean number of revertant colonies in all tested strains compared to the background spontaneous reversion rate in the negative control.
The mean number of revertant colonies in the negative controls was within the range of the historical controls.
Positive controls significantly increased the reverse mutation rate.
The results are presented in Table A6.6.1- 1and Table A6.6.1- 2.

With metabolic activation: test item dissolved in Milli-Q-water did not cause a two-fold or more increase in the mean number of revertant colonies in all tested strains compared to the background spontaneous reversion rate in the negative control.
The mean number of revertant colonies in the negative controls was within the range of the historical controls
Positive controls significantly increased the reverse mutation rate.
The results are presented in Table A6.6.1- 1 and Table A6.6.1- 2.

Cytotoxicity
Yes
In the first assay cytotoxicity was observed at and above 185 µg/plate with and without metabolic activation in all tested strains.
In the second assay, cytotoxicity was observed at 200 µg in the absence and presence of S9-mix, except for TA 1535 and TA 100 where normal growth was observed with S9 mix. At 100 µg per plate pin points were observed in TA 1537 and TA 98, in the absence of S9-mix.
The results are presented in Table A6.6.1- 1 and Table A6.6.1- 2.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table A6.6.1 -1:First assay: average numbers of revertants per plate in E.coli or S.typhimurium after treatment with the test item (plate-incorporation assay, triplicate plates per concentration).

Concentration [µg/plate]

TA 1535

TA 1537

TA 98

TA 100

E.coli

Comments

–S9

+S9

–S9

+S9

–S9

+S9

–S9

+S9

–S9

+S9

Control (mean number of revertant colonies)

26±3

28±5

23±1

17±7

47±6

69±4

191±15

167±12

33±6

37±6

 

62

27±3

21±6

16±8

23±8

27±4

67±15

162±19

173±21

34±3

40±6

 

185

17±2

8±5

11±1

107±4

30±6

37±6

Cytotoxicity

556

Cytotoxicity

1667

0

0

0

0

0

0

0

0

0

0

Cytotoxicity

5000

0

0

0

0

0

0

0

0

0

0

Cytotoxicity

Positive controls

606±42

814±114

4087±1344

288±10

2219±82

1953±83

775±58

2879±186

222±10

1527±167

 

         plate not counted due to contamination or pin points

 

Table A6.6.1-2:Second assay:average numbers of revertants per plate in E.coli or S.typhimurium after treatment with the test item (plate-incorporation assay, triplicate plates per concentration).

Concentration [µg/plate]

TA 1535

TA 1537

TA 98

TA 100

E.coli

Comments

–S9

+S9

–S9

+S9

–S9

+S9

–S9

+S9

–S9

+S9

Control (mean number of revertant colonies)

24
±9

29
±7

18
±2

26
±12

42
±15

68
±4

184
±34

173

±17

47
±10

48
±8

 

12.5

30
±7

23
±3

19
±3

27
±4

41
±8

69
±6

168
±7

162
±14

49
±6

64
±2

 

25

24
±5

18
±3

16
±3

21
±3

32
±6

52
±7

172
±13

144
±22

37
±9

44
±7

 

50

25
±3

18
±6

18
±7

29
±5

41
±2

55
±8

187
±6

179
±13

40
±4

44
±5

 

100

25
±6

23
±5

30
±4

-

51
±14

164
±37

179
±20

51
±8

47
±9

Cytotoxicity (pin points observed in TA 1537; TA 98 without S9 mix)

200

121
±4

159
±20

Cytotoxicity, (except TA 1535 and TA 100 with S9 mix)

Positive controls

721
± 36

500
± 53

2396
± 440

316
± 20

1314
± 141

845
± 107

856
± 84

2205
± 81

174
± 32

1695
± 152

 

         plate not counted due to pin points


Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

DOPA-Glycinate was not mutagenic in this bacterial reverse mutation assay in the absence and presence of metabolic activation (S9 mix).
Executive summary:

The mutagenic potential of DOPA-Glycinate (99.4% a.i.) was tested in the bacterial reverse mutation test using the plate incorporation assay. The study was conducted according to OECD guideline 471 (1997).

Two independent plate incorporation assays were performed at concentrations of up to 5000 µg per plate (first assay) or up to 200 µg/plate (second assay). In the first assay cytotoxicity was observed at ≥ 185 µg/plate ± S9 mix in all tested strains. In the second assay, cytotoxicity was observed at 200 µg ± S9 mix in all strains except for TA 1535 and TA 100.

No increase in the number of revertant colonies was found in any of the tested strains with or without metabolic activation while the positive controls gave the expected increase in the mean number of revertant colonies.

Under the conditions of this study, the test item dissolved in water was not mutagenic.