dinotefuran (ISO); 1-methyl-2-nitro-3-(tetrahydro-3-furylmethyl)guanidine

Genetic toxicity: in vitro

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 2

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

Test material information

Constituent 1

Reference substance name:

(RS)-1-methyl-2-nitro-3-(tetrahydro-3-furylmethyl)guanidine

EC Number:

605-399-0

Cas Number:

165252-70-0

Molecular formula:

C7H14N4O3

IUPAC Name:

(RS)-1-methyl-2-nitro-3-(tetrahydro-3-furylmethyl)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)
	%RS^a	RTG^b	MF^c	%RS^a	RTG^b	MF^c
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)
	%RS^a	RTG^b	MF^c	%RS^a	RTG^b	MF^c
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.