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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:
2019-03-06 to 2019-03-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

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:
1993
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Propionamide
EC Number:
201-172-1
EC Name:
Propionamide
Cas Number:
79-05-0
Molecular formula:
C3H7NO
IUPAC Name:
propanamide
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:
S9 mix induced by ß-Naphthoflavone/Phenobarbital in livers of rats. 10% and 20% S9 in the S9 mix were used in the 1st and 2nd test series, respectively.
Test concentrations with justification for top dose:
1st serie: 5, 15.8, 50, 158, 500, 1580 and 5000 µg/plate
2nd serie: 50, 158, 500, 1580 and 5000 µg/plate
The test material concentrations used were selected according to the EEC, OECD and Japanese guidelines for this test system.
Vehicle / solvent:
- Vehicle/solvent used: Ultra-pure water
- Justification for choice of solvent/vehicle: The selection of the solvent for this assay was based on the available information from a preliminary solubility test. Ultra-pure water showed best performance and was thus used for this experiment at a maximum concentration of 100 µL/plate.

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 (DAUN): -S9: TA 98; 2-Aminoanthracene (2-AA): +S9: TA 98, TA 100, TA 1535, TA 1537 and WP2 uvr A
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 3 replicates for test item concentrations and positive controls, 6 replicates for controls
- Number of independent experiments : 2

DURATION:
- Exposure duration: The incubation of plates was performed at 36 - 38 °C for 2 days.

OTHER:
-S9 concentration: 1st series 10%, 2nd series 20%
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:
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 examined
Positive controls validity:
valid
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 100
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
Additional information on results:
Following treatment with the test item, no precipitation on the agar plates occurred. No toxicity to the bacteria was observed.

Any other information on results incl. tables

Table 1: Summary 1st series

Metabolic Activation

Test material

Conc. [µg/plate]

Revertants per plate (Mean ± SD)

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

Without activation

H2O

 

38 ± 2

120 ± 11

35 ± 8

10 ± 4

36 ± 11

Test item

5.00

27 ± 8

120 ± 15

32 ± 7

6 ± 4

44 ± 7

15.8

33 ± 12

128 ± 10

33 ± 3

6 ± 2

33 ± 3

50.0

42 ± 6

119 ± 17

29 ± 7

10 ± 5

37 ± 8

158

40 ± 1

127 ± 4

36 ± 5

10 ± 2

34 ± 12

500

33 ± 6

115 ± 4

36 ± 3

13 ± 6

40 ± 14

1580

34 ± 7

119 ± 11

28 ± 13

14 ± 8

34 ± 1

5000

39 ± 2

133 ± 13

37 ± 4

9 ± 4

36 ± 1

NaN3

2.00

 

1687 ± 84

895 ± 109

 

 

NQO

2.00

 

 

 

 

386 ± 54

DAUN

5.00

565 ± 75

 

 

 

 

9-AA

50.0

 

 

 

937 ± 142

 

With activation

H2O

 

47 ± 6

143 ± 11

33 ± 7

10 ± 3

47 ± 10

Test item

5.00

34 ± 12

146 ± 2

28 ± 8

14 ± 2

48 ± 8

15.8

46 ± 3

153 ± 7

34 ± 4

10 ± 2

50 ± 7

50.0

47 ± 2

134 ± 18

36 ± 14

13 ± 3

51 ± 7

158

50 ± 3

157 ± 3

32 ± 8

9 ± 2

39 ± 5

500

58 ± 5

139 ± 18

28 ± 9

16 ± 5

45 ± 14

1580

42 ± 3

149 ± 9

36 ± 11

8 ± 4

40 ± 7

5000

48 ± 5

145 ± 12

27 ± 6

15 ± 7

40 ± 5

2-AA

10.0

 

 

 

 

729 ± 18

2-AA

2.00

594 ± 48

1074 ± 181

 

 

 

2-AA

5.00

 

 

273 ± 5

337 ± 62

 

Table 2: Summary 2nd series

Metabolic Activation

Test material

Conc. [µg/plate]

Revertants per plate (Mean ± SD)

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

Without activation

 

H2O

 

34 ± 5

132 ± 8

32 ± 5

11 ± 3

42 ± 6

Test item

5.00

34 ± 9

137 ± 8

26 ± 5

13 ± 1

42 ± 11

158

33 ± 12

126 ± 14

27 ± 6

16 ± 3

36 ± 9

500

42 ± 14

133 ± 25

29 ± 5

12 ± 4

41 ± 6

1580

27 ± 6

142 ± 13

30 ± 4

10 ± 3

39 ± 8

5000

27 ± 2

124 ± 11

34 ± 4

11 ± 0

42 ± 11

NaN3

2.00

 

1733 ± 34

941 ± 47

 

 

NQO

2.00

 

 

 

 

2212 ± 164

DAUN

5.00

406 ± 60

 

 

 

 

9-AA

50.0

 

 

 

191 ± 71

 

With activation

H2O

 

32 ± 7

172 ± 16

31 ± 6

15 ± 4

48 ± 6

Test item

5.00

33 ± 12

160 ± 21

33 ± 4

13 ± 1

48 ± 11

158

26 ± 4

166 ± 18

26 ± 3

21 ± 8

43 ± 7

500

37 ± 8

179 ± 14

27 ± 7

13 ± 2

52 ± 12

1580

30 ± 9

159 ± 16

29 ± 12

10 ± 4

47 ± 9

5000

35 ± 9

175 ± 7

26 ± 7

12 ± 4

44 ± 9

2-AA

2.00

136 ± 5

447 ± 10

 

 

 

2-AA

5.00

 

 

249 ± 8

180 ± 14

 

2-AA

10.00

 

 

 

 

248 ± 7

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions chosen, the test item did not induce gene mutations by base-pair or frameshift changes in the genome of the strains used. Therefore, it was concluded that with and without addition of S9 mix as the exogenous metabolizing system the test item was not mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation test.
Executive summary:

The mutagenic potential of the test item was investigated in a bacterial reverse mutation test according to OECD guideline 471 .
The investigations for the mutagenic potential of the test item 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 P-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.
Under the experimental conditions chosen, the test item did not induce gene mutations by base-pair or frameshift changes in the genome of the strains used. Therefore, it was concluded that with and without addition of S9 mix as the exogenous metabolizing system the test item was not mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation test.