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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27/09/2001 - 08/02/2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP, Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report date:
2002

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
1-methyl-2-nitro-3-(tetrahydrofuran-3-ylmethyl)guanidine
EC Number:
605-399-0
Cas Number:
165252-70-0
Molecular formula:
C7H14N4O3
IUPAC Name:
1-methyl-2-nitro-3-(tetrahydrofuran-3-ylmethyl)guanidine
Test material form:
solid: particulate/powder
Remarks:
powder

Method

Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Mycoplasma-free tk+/-
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Experiment 1 and 2:
0 (saline), 400, 800, 1200, 1600 and 2022 µg/mL
Both with and without metabolic activation.
See Table 1.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: saline
Controls
Untreated negative controls:
yes
Remarks:
saline diluted 10-fold in the treatment medium
Negative solvent / vehicle controls:
yes
Remarks:
as above
True negative controls:
not specified
Positive controls:
yes
Remarks:
4-nitroquinoline-1-oxide (without S9) benz(a)pyrene (with S9)
Positive control substance:
other: as above
Details on test system and experimental conditions:
Dinotefuran was dissolved in saline, under subdued lighting conditions, immediately prior to assay to give the required concentration. Stock solutions were filter-sterilised and further dilutions were made using saline. The dinotefuran solutions were protected from light and used within 2 hours of preparation. No change in osmolality occurred at the highest concentration tested.
Evaluation criteria:
The test article was considered to be mutagenic if all the following criteria were met:
• the assay was valid
• the mutant frequency at one or more doses was significantly greater than that of the negative control (p<0.05)
• there was a significant dose-relationship as indicated by the linear trend analysis (p<0.05).
Statistics:
Statistical significance of mutant frequencies (total wells with clones) was carried out according to the UKEMS guidelines. Thus the control log mutant frequency (LMF) was compared with the LMF from each treatment concentration based on Dunnett's test for multiple comparisons, and secondly the data was checked for a linear trend in mutant frequency with treatment concentration using weighted regression. The test for linear trend is one-tailed, therefore negative trend was not considered significant. These tests required the calculation of the heterogeneity factore to obtain a modified estimate of variance.

Results and discussion

Test results
Key result
Species / strain:
mouse lymphoma L5178Y cells
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:
Genotoxicity without metabolic activation:

Experiment 1:
No: relative survival at 2022µg/mL was 102.68 without activation.

Experiment 2:
No: relative survival at 2022µg/mL was 76.16 without metabolic activation.

See Table 3

Genotoxicity with metabolic activation:

Experiment 1:
No: relative survival at 2022µg/mL was 118.06% with metabolic activation.

Experiment 2:
No: relative survival at 2022µg/mL was 100.28% with metabolic activation.

See Table 3

Cytotoxicity:

No: no statistically significant increases in mutation frequency occurred at any dose level in the absence or presence of metabolic activation in either independent experiment. The proportion of small colony mutants for the solvent controls without and with metabolic activation ranged from 38 to 41% in experiment 1 and from 52 to 55% in experiment 2. Marked increases in the numbers of both small and large colony mutants occurred in response to both positive control materials.

Analysis of dinotefuran formulations demonstrated achieved concentrations within 100 ± 10% of nominal concentrations. The assay acceptance criteria were met and the study is considered valid.

See Table 2.

Remarks on result:
other: all strains/cell types tested

Any other information on results incl. tables

Table 1: Dose levels in two preliminary studies and main assay

Study

Dose levels ofdinotefuranused (µg/mL)

Cytotoxicity ± S9, 3h

62.5

125

250

500

1000

2022

 

 

 

Cytotoxicity-S9, 24h

7.81

15.63

31.25

62.5

125

250

500

1000

2022

Experiment 1 in main assay ± S9, 3h

400

800

1200

1600

2022

 

 

 

 

Experiment 2 inmain assay-S9, 24h; +S9, 3h

400

800

1200

1600

2022

 

 

 

 

 

Table 2: Summary of relative survival in the cytotoxicity range-finding study

Compound

Dose level

Relative survival (%):

 

(µg/mL)

3-hour exposure (-S9)

3-hour exposure (+S9)

24-hour exposure (-S9)

DMSO

0

= 100

= 100

= 100

Dinotefuran

7.81

-

-

150.17

 

15.63

-

-

122.63

 

31.25

-

-

116.22

 

62.5

47.60

58.55

101.05

 

125

103.20

72.40

111.20

 

250

97.81

82.08

109.29

 

500

93.43

94.94

133.42

 

1000

132.27

66.25

97.63

 

2022

87.00

70.00

109.58

- not assayed


Table 3: Relative survival and mutant frequency – main experiments

Concn.

Experiment 1

(mg/mL)

3-hr exposure (-S9)

3-hr exposure (+S9)

 

%RSa

RTGb

MFc

%RSa

RTGb

MFc

0

= 100

1.00

67.92

= 100

1.00

81.65

400

100.42

0.87

70.05

103.94

1.11

80.01

800

98.63

1.06

70.70

116.66

1.18

54.15

1200

112.29

1.12

60.56

105.90

1.12

64.32

1600

103.08

0.99

85.24

105.69

1.14

68.82

2022

102.68

0.91

81.84

118.06

1.03

61.05

NQO 0.05

0.01

0.02

0.04

 

102.70

94.13

-

-

 

0.85

0.54

-

-

 

149.24

337.64

-

-

 

-

-

-

-

 

-

-

-

-

 

-

-

-

-

BP

2.0

3.0

 

-

-

 

-

-

 

-

-

 

91.27

81.81

 

0.76

0.69

 

365.23

525.10

 

Experiment 2

 

24-hr exposure (-S9)

3-hr exposure (+S9)

 

%RSa

RTGb

MFc

%RSa

RTGb

MFc

0

= 100

1.00

72.69

= 100

 

101.87

400

96.10

1.04

61.05

105.89

 

78.81

800

88.13

0.99

62.01

112.71

 

82.82

1200

83.95

1.09

85.11

116.11

 

129.05

1600

107.55

1.01

56.23

101.87

 

105.00

2022

76.16

0.94

82.33

100.28

 

107.68

NQO 0.05

0.01

0.02

0.04

 

-

-

61.06

55.07

 

-

-

0.84

0.92

 

-

-

194.58

196.02

 

-

-

-

-

 

-

-

-

-

 

-

-

-

-

BP

2.0

3.0

 

-

-

 

-

-

 

-

-

 

102.04

62.54

 

0.58

0.41

 

423.87

606.07

a% relative survival adjusted by post-treatment cell count;

brelative total growth;

c5-TFT resistant mutants/106viable cells 2 days after treatment;

- not assayed

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results :negative

In both experiments relative survival at 2022µg/mL was at least 76.16 % with and without metabolic activation. No statistically significant increases in mutation frequency occurred at any dose level in the absence or presence of metabolic activation in either independent experiment. Marked increases in the numbers of both small and large colony mutants occurred in response to both positive control materials.

It was concluded that dinotefuran technical and/or metabolites does not induce mutation at the tk locus of L5178Y mouse lymphoma cells at concentrations up to 10mM, and is considered not mutagenic in this test system.