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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 September 2017 - 06 December 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
df. Sofuni, 1993
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium pyruvate
EC Number:
204-024-4
EC Name:
Sodium pyruvate
Cas Number:
113-24-6
Molecular formula:
C3H4O3.Na
IUPAC Name:
sodium 2-oxopropanoate
Test material form:
solid

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
beta-naphthoflavone/phenobarbital induced rat liver S9 mix
Test concentrations with justification for top dose:
test concentrations: 5, 15.8, 50, 158, 500, 1580 and 5000 µg/plate
top dose: maximum recommended concentration according to current regulatory guideline (OECD 471, 1997)
Vehicle / solvent:
- Vehicle/solvent used: ultrapure water
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
sodium azide
other: daunomycin: 1 µg/plate, 2-aminoanthracene: 2-10 µg/plate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 2 days

NUMBER OF REPLICATIONS: 3 replicates for test item concentrations and positive controls, 6 replicates for solvent controls

DETERMINATION OF CYTOTOXICITY
- Method: counting numbers of revertants

OTHER:
-S9 concentration: 1st series 10%, 2nd series 30%
Evaluation criteria:
A test material was to be defined as positive or mutagenic in this assay if
- the assay is considered valid and
- a biologically relevant increase in the mean number of revertants above a threshold of 2-fold (TA 98, TA 100, WP2 uvrA) or 3-fold (TA 1535, TA 1537) as compared to the concurrent negative controls is observed
- an increase exceeding the threshold at only one concentration is considered as biologically meaningful if reproduced in a second independent experiment
- a concentration-dependent increase is considered biologically meaningful if the threshold is exceeded at more than one concentration
A test material is defined as negative or non-mutagenic in this assay if
- the assay is considered valid and
- none of the above mentioned criteria are met

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
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
Key result
Species / strain:
S. typhimurium TA 1537
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
Key result
Species / strain:
S. typhimurium TA 98
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
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation occurred.

Any other information on results incl. tables

Table 1: Summary 1st series

Metabolic Activation

Test Material

Concentr. [µg/plate]

Revertants per plate (Mean ± SD)

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

Without Activation

H2O

 

31 ± 8

113 ± 8

24 ± 5

10 ± 3

39 ± 16

Test item

5.00

30 ± 10

110 ± 3

24 ± 6

9 ± 2

35 ± 5

15.8

35 ± 9

106 ± 17

30 ± 10

7 ± 1

33 ± 5

50.0

31 ± 5

128 ± 16

28 ± 3

8 ± 2

34 ± 5

158

38 ± 4

125 ± 17

23 ± 1

12 ± 3

32 ± 7

500

26 ± 1

118 ± 12

24 ± 1

10 ± 1

37 ± 3

1580

31 ± 5

125 ± 30

26 ± 5

6 ± 1

33 ± 4

5000

30 ± 2

115 ± 8

24 ± 9

7 ± 1

36 ± 1

DAUN

1.00

405 ± 22

 

 

 

 

NaN3

2.00

 

1830 ± 110

993 ± 52

 

 

9-AA

50.0

 

 

 

1376 ± 505

 

NQO

2.00

 

 

 

 

1491 ± 231

With Activation

H2O

 

41 ± 9

117 ± 17

22 ± 4

11 ± 4

41 ± 5

Test item

5.00

33 ± 2

106 ± 10

17 ± 4

10 ± 5

36 ± 7

15.8

41 ± 4

118 ± 15

12 ± 3

8 ± 2

41 ± 4

50.0

33 ± 5

119 ± 31

20 ± 8

10 ± 3

41 ± 7

158

32 ± 5

107 ± 24

16 ± 3

12 ± 5

43 ± 9

500

35 ± 7

109 ± 6

24 ± 3

7 ± 7

32 ± 8

1580

29 ± 6

110 ± 14

18 ± 6

11 ± 2

35 ± 5

5000

28 ± 3

114 ± 7

18 ± 3

7 ± 2

39 ± 5

2-AA

2.00

827 ± 59

1511 ± 150

 

 

 

2-AA

5.00

 

 

154 ± 12

397 ± 28

 

2-AA

10.0

 

 

 

 

489 ± 9

Key to Positive Controls: NaN3: Sodium azide, 2-AA: 2-Aminoanthracene, 9-AA: 9-Aminoacridine, DAUN: Daunomycin, NQO: 4-Nitroquinoline-N-oxide

 

Table 2: Summary 2nd series

Metabolic Activation

Test Material

Concentr. [µg/plate]

Revertants per plate (Mean ± SD)

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

Without Activation

H2O

 

36 ± 5

102 ± 16

18 ± 3

6 ± 3

34 ± 6

Test item

50.0

37 ± 10

94 ± 6

22 ± 6

9 ± 1

29 ± 3

158

31 ± 8

108 ± 10

25 ± 5

5 ± 2

33 ± 7

500

24 ± 6

100 ± 15

25 ± 4

7 ± 1

39 ± 12

1580

27 ± 5

101 ± 4

20 ± 1

5 ± 2

31 ± 2

5000

35 ± 3

104 ± 17

15 ± 4

8 ± 3

30 ± 4

DAUN

1.00

199 ± 9

 

 

 

 

NaN3

2.00

 

1592 ± 80

914 ± 42

 

 

9-AA

50.0

 

 

 

820 ± 212

 

NQO

2.00

 

 

 

 

2085 ± 77

With Activation

H2O

 

42 ± 8

98 ± 12

18 ± 8

11 ± 3

41 ± 10

Test item

50.0

39 ± 13

102 ± 9

18 ± 5

10 ± 3

32 ± 14

158

39 ± 8

96 ± 6

16 ± 2

10 ± 4

38 ± 5

500

36 ± 1

93 ± 26

16 ± 5

15 ± 7

44 ± 1

1580

36 ± 2

95 ± 3

12 ± 4

7 ± 3

34 ± 4

5000

36 ± 3

94 ± 7

14 ± 3

9 ± 4

43 ± 12

2-AA

2.00

439 ± 96

784 ± 255

 

 

 

2-AA

5.00

 

 

132 ± 18

138 ± 81

 

2-AA

10.0

 

 

 

 

232 ± 48

Key to Positive Controls: NaN3: Sodium azide, 2-AA: 2-Aminoanthracene, 9-AA: 9-Aminoacridine, DAUN: Daunomycin, NQO: 4-Nitroquinoline-N-oxide

 

 

Applicant's summary and conclusion

Conclusions:
The test item is considered non-mutagenic under the test conditions described.
Executive summary:

The present study was conducted to investigate the test material for its mutagenic potential in a bacterial reverse gene mutation assay in the absence and presence of a rat liver metabolizing system (S9 mix). The investigations were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 1535 and TA 1537 and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from rats pretreated with beta-Naphthoflavone/Phenobarbital was used. In this study, two experimental series were performed. The S9 mix used contained 10% S9 in the 1st and 20% S9 in the 2nd series, respectively. Solvent and positive control treatments were included for all strains. The mean numbers of revertant colonies were all within acceptable ranges for solvent control treatments, or were clearly elevated by positive control treatments, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used. Following treatment of all bacteria tester strains with the test item in the absence and presence of S9 mix, no relevant increases in revertant numbers were observed. It was concluded that with and without addition of S9 mix as the exogenous metabolizing system, the test item was not mutagenic under the experimental conditions described.