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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:
18 July 2017 to 27 July 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:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
"Standards of Mutagenicity Tests using Microorganisms" (Notification No. 77, September 1 1988 & partial revision: Notification No. 67, June 2, 1997, Ministry of Labour, Japan and Notification No. 120, December 25, 2000, Ministry of Labour, Japan) and the "Amendment of the Reporting Form of the Results of the Mutagenicity Tests using Microorganims" (Notification No. 653, September 29, 1997, Labour Standards Bureau, Ministry of Labour, Japan)
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
Guidelines of Toxicity Testing of New Chemical substances (Notification No.7 of 0331 Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare, No. 5 of the Manufacturing Industries Bureau, Ministry of Economy and Industry, & No. 110331009 of Environmental Policy Bureau, Ministry of the Environment, Japan, 31 March 2011)
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2,4-dihydro-2,5-dimethyl-3H-pyrazol-3-one
EC Number:
220-389-2
EC Name:
2,4-dihydro-2,5-dimethyl-3H-pyrazol-3-one
Cas Number:
2749-59-9
Molecular formula:
C5H8N2O
IUPAC Name:
1,3-dimethyl-4,5-dihydro-1H-pyrazol-5-one
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot No.of test material: 7E18
- Expiration date of the lot: 14 Sept. 2017
- Purity: 99.98%
- Name and concentration of impurities: Unknown: 0.02%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

OTHER SPECIFICS:
- Appearance: White powder

Method

Target gene:
his- (S. typhimurium)
trp- (E. coli)
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital and 5,6-benzoflavone induced rat liver S9 mix
Test concentrations with justification for top dose:
Dose levels in the preliminary test (with and without S9 mix, all strains): 1.2, 4.9, 20, 78, 313, 1250 and 5000 μg/plate.

Dose levels in the 1st main test and 2nd main test
- without S9 mix, TA 100, TA 1535: 10, 20, 39, 78, 156, 313, 625, 1250, 2500, 5000 μg/plate
- without S9 mix, TA 98: 39, 78, 156, 313, 625, 1250 µg/plate
- without S9 mix, TA 1537: 10, 20, 39, 78, 156, 313 µg/plate
- without S9 mix, WP2 uvrA: 313, 625, 1250, 2500, 5000 µg/plate
- with S9 mix, all strains: 313, 625, 1250, 2500, 5000 µg/plate

Justification for the top dose:
The top doses were selected on the basis on the preliminary test in which, the growth inhibition by the test substance was observed at 313 μg/plate and more in TA100, TA1535 and TA1537 without metabolic activation, and at 1250 μg/plate and more in TA98 without metabolic activation. And an increase in the number of revertant colonies more than twice in comparison with that of the negative control was observed in TA100 and TA1535 without metabolic activation. And the precipitate of the test substance on the plates was not observed either with or without metabolic activation. Therefore, as the highest dose level of the test substance in the main tests, the 313 μg/plate dose was selected for TA1537 without metabolic activation, and the 1250 μg/plate dose was selected for TA98 without metabolic activation, and these highest doses were diluted 5 times (using a common ratio of 2) to provide a total of 6 dose levels. And the 5000 μg/plate dose was selected for WP2 uvrA without metabolic activation and all strains with metabolic activation, and this highest dose was diluted 4 times (using a common ratio of 2) to provide a total of 5 dose levels. Moreover, in order to confirm dose-related response, the 5000 µg/plate dose was selected for TA100 and TA1535 without metabolic activation, and this highest dose was diluted 9 times (using a common ratio of 2) to provide a total of 10 dose levels.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Water for injection (The Japanese Pharmacopoeia)
- Justification for choice of solvent/vehicle: Based on the information from the sponsor, the test substance was soluble at 1690 g/L and stable in water. Therefore water for injection was used as solvent for preparation.
Controls
Untreated negative controls:
yes
Remarks:
sterility
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2), 2-Methoxy-6-chloro-943-(2-chloroethypaminopropylamino]acridine • 2HCl (ICR-191), 2-Aminoanthracene (2AA)
Remarks:
Positive controls were chosen in accordance with the "Standards for Mutagenicity Tests using Microorganisms," the "Guidelines for Toxicity Testings of New Chemical Substances" and the "OECD GUIDELINE FOR THE TESTING OF CHEMICALS 471."
Details on test system and experimental conditions:
METHOD OF APPLICATION OF 1st and 2nd MAIN TEST: in medium

DURATION
- Preincubation period: at 37°C for 20 minutes
- Exposure duration: at 37°C for 48 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method:growth inhibition of the test strains was checked using a stereoscopic microscope
- Any supplementary information relevant to cytotoxicity: Sterility test was performed
Evaluation criteria:
In the two main tests, if the number of revertant colonies on the test plates increased significantly in comparison with that on the control plates (based on twice as many as that of the negative control), and dose-response and reproducibility were also observed, the test substance was to be judged positive. The results at each concentration were demonstrated with the mean and the standard deviation.
Statistics:
Not specified. Mean and standard deviations of the test results were calculated

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: TA 100, TA 98, TA 1537
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: TA 100, TA 98, TA 1537
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
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:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
The precipitate of the test substnace on the plates was not observed iether with or without S9 mix.

PRELIMINARY TEST
An increase in the number of revertant colonies was observed in TA100 without S9 mix, and increased more than twice of the negative control

POSITIVE CONTROLS
The revertant colonies of the positive controls showed an increase of more than twice that of the negative controls and they were within limit of control (mean ± 3SD) in historical data, indicating that this study was performed correctly.

CYTOXICITY
The growth inhitition of the test strains by the test substance was observed in different strains at different dosages. See the 2 Tables in the "any other information on results incl. tables" for more information.
Remarks on result:
other: 1st and 2nd main test

Any other information on results incl. tables

Table of the 1st main test results

 

Base-pair substitution type

Frame-shift type

S9 mix

Dose μg/plate

TA 100

TA 1535

WP2 uvrA

TA98

TA1537

 

Solvent control

102 ± 5.7 *

15 ± 1.7

29 ± 4.0

25 ± 3.2

9 ± 1.2

Without S9

10

99 ± 3.5

13 ± 2.0

NT

NT

9 ± 15

20

111 ± 21.1

16 ± 2.1

NT

NT

7 ± 1.0

39

100 ± 3.8

12 ± 1.0

NT

24± 5.5

10 ± 1.2

78

106 ± 3.1

14 ± 2.5

NT

23 ± 3.1

8 ± 1.5

156

112 ± 13.1

17 ± 4.0

NT

24 ± 4.6

10 ± 0.6

313

138 ± 13.5 *

14 ± 1.2 *

24 ± 6.1

25 ± 4.9

10 ± 2.6 *

625

176 ±17.4 *

16 ± 2.1 *

20 ± 1.0

23 ± 4.0

NT

1250

181 ± 12.8 *

46 ± 7.0 *

25 ± 3.5

27 ± 4.0 *

NT

2500

154 ± 23.3 *

48 ± 3.0 *

24 ± 2.6

NT

NT

5000

117 ± 15.0 *

20 ± 1.7 *

21 ± 3.2

NT

NT

 

 

 

 

 

 

 

With S9

Solvent control

108 ± 12.2

15± 2.1

28 ± 3.8

35 ± 3.6

15 ± 1.7

313

105 ± 2.9

14 ± 3.5

24 ± 1.5

31 ± 6.4

15 ± 2.1

625

113 ± 17.6

18 ± 1.0

24 ± 2.6

29 ± 0.6

13 ± 1.0

1250

109 ± 13.9

17 ± 0.6

26 ± 4.5

29 ± 1.5

13 ± 1.2

2500

98 ± 6.7

17 ± 2.6

24 ± 0.6

29 ± 3.2

13 ± 1.0

5000

147 ± 8.1

14 ± 3.0

23 ± 1.0

26 ± 1.2

13 ± 1.0

NT not tested

* = The growth inhibition of the tested bacterium by the test substance was observed

Table of the 2nd main test results

 

Base-pair substitution type

Frame-shift type

S9 mix

Dose μg/plate

TA100

TA 1535

WP2 uvrA

TA98

TA1537

 

Solvent control

91 ± 8.5

12 ± 1.5

24 ± 2.6

33 ± 3.1

10 ± 1.2

Without S9

10

101 ± 9.3

11 ± 2.6

NT

NT

7 ± 0.0

20

90 ± 10.4

11 ± 1.5

NT

NT

9 ± 2.0

39

92 ± 3.2

11 ± 2.5

NT

28 ± 2.0

9 ± 1.5

78

97 ± 13.6

9 ± 0.6

NT

30 ± 3.1

7 ± 1.0

156

111 ± 12.3

11 ± 2.6

NT

30 ± 2.6

8 ± 2.1

313

134 ± 12.5 *

16 ± 1.5 *

24 ± 1.7

31 ± 1.2

9 ± 2.5 *

625

143 ± 11.5 *

15 ± 2.3 *

26 ± 2.5

25 ± 4.9

NT

1250

153 ± 9.8 *

29 ± 3.0 *

22 ± 2.0

30 ± 5.3 *

NT

2500

153 ± 9.8 *

49 ± 6.2 *

23 ± 5.2

NT

NT

5000

98 ± 12.4 *

11 ± 1.2 *

26 ± 3.8

NT

NT

 

 

 

 

 

 

 

With S9

Solvent control

119 ± 18.0

12 ± 1.5

32 ± 1.0

37 ± 4.6

18 ± 1.0

313

96 ± 6.4

10 ± 2.0

29 ± 5.8

35 ± 3.5

19 ± 1.5

625

108 ± 9.3

10 ± 2.1

30 ± 1.0

33 ± 3.0

17 ± 1.7

1250

107 ± 14.8

14 ± 3.1

31 ± 2.6

38 ± 3.6

17 ± 2.5

2500

143 ± 7.4

12 ± 1.5

32 ± 2.0

33 ± 4.9

16 ± 2.6

5000

147± 9.1

10 ± 0.6

38 ± 2.0

33 ± 6.2

15 ± 3.2

NT not tested

* = The growth inhibition of the tested bacterium by the test substance was observed

Applicant's summary and conclusion

Conclusions:
The test item is considered to be mutagenic to bacteria under the described study conditions.
Executive summary:

In this GLP compliant study, performed according to OECD guideline 471, the mutagenicity potential of the test item was assessed with Salmonella typhimurium TA100, TA1535, TA98, TA1537 and Escherichia coli WP2 uvrA using the bacterial reverse mutation assay. The test item was tested in doses of 10, 20, 39, 78, 156, 313, 625, 1250, 2500, 5000 µg/plate using with/without metabolic activation. For metabolic activation S9 -mix retrieved from phenobarbital and 5.6 -benzoflavone induced rat livers. In this study, a dose-dependent increase in the number of revertant colonies more than twice in comparison with that of the negative control and reproducibility were observed in S. typhimurium TA1535 without metabolic activation. The positive controls and negative controls tested showed that the study was performed correctly. From the above, it is therefore judged that the test substance is mutagenic under the described study conditions.

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