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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Four in vitro genotoxicity studies were negative for genotoxicity endpoints with and without metabolic activation. Positive control substances in these studies induced positive effects.

Link to relevant study records

Reference

Reference 1

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

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

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

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.

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

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

 

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.

Reference 2

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12/06/1996 - 10/07/1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP, Guideline study

Qualifier:

according to guideline

Guideline:

other: JMAFF 59 NohSan no. 4200 (1985)

Deviations:

no

GLP compliance:

yes

Type of assay:

other: Unscheduled DNA synthesis in mammalian cells in vitro

Species / strain / cell type:

bacteria, other: Bacillus subtilis: M45 Rec- and H17 Rec+

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

0, 62.5, 250, 1000, 4000 and 16000µg/disc dinotefuran with and without metabolic activation.

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Remarks:

Kanamycin sulfate (without S9); Streptomycin sulfate (with S9)

Negative solvent / vehicle controls:

yes

Remarks:

As above

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: Mitomycin C (without S9); Trp-P-1 (with S9)

Details on test system and experimental conditions:

An 8mm paper disc (2 discs/dose) was treated with 20 µL solvent or test substance solution and placed on the spore-innoculated agar without metabolic activation. With metabolic activation, an 8mm paper disc (2 discs/dose level) was treated with 20 µL coenzyme solution and 20 µL solvent or test substance solution and placed on the spore-innoculated agar containing S9 mix.

Evaluation criteria:

Results are judged positive when the difference in diameter of growth inhibition zones between strains H17 and M45 is more than 5mm, and negative if less than 2.5mm. Where differences in inhibition zone diameter are between 2.5 and 5.0mm the results are evaluated by consideration of dose-response and reproducibility.

Statistics:

Not reported

Key result

Species / strain:

other: Bacillus subtilis: M45 Rec- and H17 Rec+

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:

Preliminary study with and without metabolic activation:
No growth inhibition occurred at any dose level in either strain with or without metabolic activation (Table 1).
Based on these results, dinotefuran in DMSO solvent was tested in H17 and M45 (approx. 2 x 107cells/mL) at concentrations of 0 (DMSO only), 1000, 2000, 4000, 8000 and 16000µg/disc with and without S9 metabolic activation.

Main study with and without metabolic activation:
DMSO solvent alone and all doses of DINOTEFURAN employed, up to and including the highest dose, 16000µg/disc, produced no growth inhibition of either strain of B. subtilis either with or without S9 metabolic activation (Table 2). The negative control substances, KM without S9 and SM with S9 produced differences in the inhibition zone diameters of 1.7 and 1.1mm, respectively, whereas the positive control substances, MMC without S9 and Trp-P-1 with S9, produced differences of 7.2 and 6.0mm, respectively.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1:Summary of growth inhibition for the dose range-finding study

Compound	Dose level	± S9	Growth inhibition zone (mm):
	(µg/disc)		M45 (Rec-)	H17 (Rec+)	Difference (M45 –H17)
DMSO	0	-	0	0	0
Dinotefuran	1000	-	0	0	0
	2000	-	0	0	0
	4000	-	0	0	0
	8000	-	0	0	0
	16000	-	0	0	0
KM	10	-	10.0	8.4	1.6
MMC	0.01	-	6.9	0	6.9
DMSO	0	+	0	0	0
Dinotefuran	1000	+	0	0	0
	2000	+	0	0	0
	4000	+	0	0	0
	8000	+	0	0	0
	16000	+	0	0	0
SM	100	+	3.3	2.7	0.6
Trp-P-1	3	+	5.6	0	5.6

 

Table 2:Summary of growth inhibition for the main assay

Compound	Dose level	± S9	Growth inhibition zone (mm):
	(µg/disc)		M45 (Rec-)	H17 (Rec+)	Difference (M45 – H17)
DMSO	0	-	0	0	0
Dinotefuran	1000	-	0	0	0
	2000	-	0	0	0
	4000	-	0	0	0
	8000	-	0	0	0
	16000	-	0	0	0
KM	10	-	10.5	8.8	1.7
MMC	0.01	-	7.2	0	7.2
DMSO	0	+	0	0	0
Dinotefuran	1000	+	0	0	0
	2000	+	0	0	0
	4000	+	0	0	0
	8000	+	0	0	0
	16000	+	0	0	0
SM	100	+	4.0	2.9	1.1
Trp-P-1	3	+	6.0	0	6.0

 

Conclusions:

Interpretation of results :
negative

It was concluded that under the conditions of the study, dinotefuran and/or metabolites does not exhibit DNA-damaging activity in B. subtilis under the conditions of the study at doses up to 16000µg/disc

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14/06/1996 - 15/10/1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP, Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF 59 NohSan no. 4200 (1985)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMHW, Part 1 (1990); Japan Ministry of Labor, Appendix 1, Notification nos. 143 (1987)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

other: Chinese hamster lung (CHL/IU)

Details on mammalian cell type (if applicable):

Mycoplasma-free

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

2000 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: saline for direct method and S9 method.

Untreated negative controls:

yes

Remarks:

saline

Negative solvent / vehicle controls:

yes

Remarks:

as above

True negative controls:

not specified

Positive controls:

yes

Remarks:

Mitomycin C (without S9) Cyclophosphamide (with S9)

Positive control substance:

other: as above

Details on test system and experimental conditions:

Dinotefuran was dissolved in saline. For preparation of the original solution and serial dilution of dinotefuran, the solution in 11-fold of each final concentration was prepared. In the cell growth inhibition test, 101.6 mg of dinotefuran dissolved in 4.6 mL of saline (final concentration is 2000 mg/mL which was equal to 10 mM) both with and without S9. In the main study, 146.3 mg of of dinotefuran dissolved in 6.65 mL of saline without S9 and 86.8 mg of dinotefuran dissolved in 3.9 mL of saline with S9 (final concentration is 2000 mg/mL which was equal to 10 mM).

Mitomycin C (MMC - 0.03µg/mL) was the positive control material for 24 and 48 hour exposures without activation and cyclophosphamide (CP - 12µg/mL) for 6 hour exposures with and without activation.

Evaluation criteria:

The test substance was to be considered as, negative (-) if the total incidence of structural aberrant cells (including gaps)or numberical aberration cells obtained from 2 dishes (total 200 metaphase) was less than 5%, suspected positive (±) if it was more than 5% and less than 10%, and positive (+) if it was more than 10%.

Statistics:

Not conducted

Species / strain:

other: Chinese hamster lung (CHL/IU)

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: No.
Structural or numerical aberrations were not induced after 6 hours exposure to dinotefuran either in the absence of metabolic activation. Cell survival was unaffected at all dose levels after 6 hours exposure.

MMC in the absence of metabolic activation produced marked increases in the incidences of structural aberrations, notably chromatid breaks and exchanges.

See Table 1 and Table 2.

Genotoxicity with metabolic activation:

No. Structural or numerical aberrations were not induced after 6 hours exposure to dinotefuran either in the presence of metabolic activation. Cell survival was unaffected at all dose levels after 6 hours exposure.

Cyclophosphamide in the presence of metabolic activation produced marked increases in the incidences of structural aberrations, notably chromatid breaks and exchanges.

See Table 1 and Table 2.

Cytotoxicity:
No. In the main study, structural or numerical aberrations were not induced by dinotefuran either after 24 or 48 hours exposure to dose levels up to 2000 µg/mL. However, slight growth inhibition occurred at 2000 µg/mL, 28% after 24 hours and 38% after 48 hours exposure.

See Table 1 and Table 2.

Remarks on result:

other: all strains/cell types tested

Table 1: Cell growth rate (%) relative to solvent control

Dose level	Preliminary study exposed for:		Main study exposed for:
(µg/mL)	48hr (-S9)	6hr (+S9)	24hr (-S9)	48hr (-S9)	6hr (+S9)	6hr (-S9)
0	=100	=100	=100	=100	=100	=100
7.81	99	100	-	-	-	-
15.6	88	93	-	-	-	-
31.3	96	102	-	-	-	-
62.5	93	88	-	-	-	-
125	92	85	-	-	-	-
250	92	85	-	-	-	-
500	88	90	85	93	100	103
1000	82	88	84	95	99	104
2000	65	100	72	62	104	106
MMC 0.03 CP 12.0	- -	- -	70 -	74 -	- 86	- 99

 

Table 2: Summary of results from the main assay

Compound	Dose	Cell survival	No. cells	No. cells with structural aberrations:						Total less	Polyploidy
	(µg/mL)	(%)		gap	ctb	cte	csb	cse	other	gaps (%)	(%)
		6-hour exposure, without S9
Saline	0	100	200	0.5	0	0	0	0	0	0.5	0.5
Dinotefuran	500	103	200	0	0	0.5	0	0	0	0.5	1.0
	1000	104	200	0.5	0	0	0	0	0	0.5	0
	2000	106	200	0	0.5	0.5	0	0	0	1.0	1.0
CP	12.0	99	200	1.0	0	0	0.5	0	0	1.5	0.5
		6-hour exposure with S9
Saline	0	100	200	0	0.5	0	0	0	0	0.5	1.0
Dinotefuran	500	100	200	0	0.5	0	0	1.0	0	1.5	1.0
	1000	99	200	0	0.5	0	0	0	0	0.5	2.5
	2000	104	200	0	0	0	0	1.0	0	1.0	1.0
CP	12.0	86	200	0	15.5	43.5	0	0.5	0	51.0	1.0
		24-hour exposure, without S9
Saline	0	100	200	0.5	0.5	0	0	1.5	0	2.5	0
Dinotefuran	500	85	200	0	2.5	2.0	0	0	0	4.5	0
	1000	84	200	0.5	1.0	1.0	0	0	0	2.5	0.5
	2000	72	200	0.5	2.0	0.5	0.5	0	0	3.5	0
MMC	0.03	70	200	2.0	42.5	38.5	1.0	0.5	0	65.5	0.5
		48-hour exposure, without S9
Saline	0	100	200	1.0	1.5	0	0.5	0	0	3.0	2.5
Dinotefuran	500	93	200	0	1.0	0.5	0	0.5	0	2.0	0.5
	1000	95	200	1.0	0	0	0	1.0	0	2.0	0.5
	2000	62	200	0	1.0	0	0	0	0	1.0	1.5
MMC	0.03	74	200	2.0	36.0	66.5	0	0.5	0	76.0	0.5

ctb-chromatid break;

cte-chromatid exchange;

csb-chromosome break;

cse-chromosome exchange including dicentric and ring chromosomes;

other-otheraberrations including fragmentation;

gap - includes both chromatid and chromosome gaps

Conclusions:

Interpretation of results: negative

In the main study, structural or numerical aberrations were not induced by dinotefuran either after 24 or 48 hours exposure to dose levels up to 2000µg/mL. However, slight growth inhibition occurred at 2000µg/mL, 28% after 24 hours and 38% after 48 hours exposure. Similarly, structural or numerical aberrations were not induced after 6 hours exposure to dinotefuran either in the presence or absence of metabolic activation. Cell survival was unaffected at all dose levels after 6 hours exposure.

It was concluded that under the conditions of this study dinotefuran and/or metabolites does not induce structural or numerical chromosomal aberrations in Chinese hamster lung cells at dose levels up to and including 2000µg/mL (equivalent to 10 mM).

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12/07/1996 - 02/10/1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP, Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1994

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Version / remarks:

1994

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU Method B.14

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Deviations:

no

Principles of method if other than guideline:

Deviations: Yes, in addition to the required 4 S. typhimurium strains, one strain of E. coli was also included since this is a requirement for Japanese authorities. The deviation does not affect the validity of the study.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

S. typhimurium: histidine-auxotrophic strains

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

E. coli: tryptophan-auxotrophic strain

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Pre-incubation: 0 (solvent control), 313, 625, 1250, 2500 and 5000g/plate
Main assay: 0 (solvent control), 1.2, 4.9, 20, 78, 313, 1250 and 5000 µg/plate dinotefuran and the relevant positive controls both with and without metabolic activation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

Remarks:

Same as negative control

True negative controls:

not specified

Positive controls:

yes

Remarks:

Without S9: 2-(2-furyl)-3-(5-nitro-2-furyl)-acrylamide (AF-2), 9-Aminoacridine and Sodium azide With S9: 2-Aminoanthracene. See Table 1.

Positive control substance:

other: See above positive controls

Details on test system and experimental conditions:

METHOD OF APPLICATION: dissolved in medium
DURATION
- Preincubation period: 20 minutes at 37°C

Evaluation criteria:

Evaluated for colonies by an automatic counter and for precipitation and growth inhibition of the background lawn by microscopy.

Dinotefuran was considered positive if at least one dose produced a mean reversion frequency in a given strains that was at least two times greater than that of the corresponding negative control plates, and the response was dose-dependent and reproducibility were observed.

Statistics:

Not reported

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Dinotefuran did not influence the growth of any strain tested at dose levels of up to 5000µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Genotoxicity without metabolic activation:

Dose range-finding study:
No, normal background growth occurred in all strains without metabolic activation. None of the strains showed an appreciable increase in the reversion frequency at any of the dose levels tested.

Main study:
No appreciable increase in the reversion frequencies occurred in any strain tested in the dose range 313 - 5000µg/plate.
See Table 2 and Table 3.

Genotoxicity with metabolic activation:

Dose range-finding study:
No, normal background growth occurred in all strains with metabolic activation. None of the strains showed an appreciable increase in the reversion frequency at any of the dose levels tested.

Main study:
No appreciable increase in the reversion frequencies occurred in any strain tested in the dose range 313 - 5000µg/plate.
See Table 2 and Table 3.

Cytotoxicity:

Dose range-finding study:
No appreciable cytotoxicity was observed at up to 5000µg/plate, the highest concentration evaluated.

Main study:
Dinotefuran did not influence the growth of any strain tested at dose levels of up to 5000µg/plate.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1: Strain specific positive control substances and dose levels employed

Strain	Without S9		With S9
	Positive control	Dose (µg/plate)	Positive control	Dose (µg/plate)
S. typhimurium TA100	AF-2	0.01	2-AA	1.0
S. typhimurium TA98	AF-2	0.01	2-AA	2.0
S. typhimurium TA1535	NaN3	0.5	2-AA	2.0
S. typhimurium TA1537	9-AA	80.0	2-AA	1.5
E. coli WP2uvrA	AF-2	0.01	2-AA	10.0

AF-2:2-(2-furyl)-3-(5-nitro-2-furyl)-acrylamide;NaN3: sodium azide; 9-AA:9-Aminoacridine;

2-AA:-Aminoanthracene

 

Table 2: Summary of the incidences of revertant colonies for the dose range-finding assay

Treatment	Dose	Mean (n = 3) ± SD revertant colonies/plate for strain:
	(µg/plate)	TA100	TA1535	WP2uvrA	TA98	TA1537
		Without S9
DMSO	0	119	7	14	11	3
Dinotefuran	1.2	138	4	17	9	4
	4.9	128	5	13	13	3
	20	129	9	17	10	4
	78	129	6	16	10	2
	313	134	7	13	8	4
	1250	122	6	15	5	4
	5000	124	6	17	9	3
AF-2	0.01	829	-	-	-	-
NaN3	0.5	-	180	-	-	-
AF-2	0.01	-	-	104	-	-
AF-2	0.1	-	-	-	475	-
9-AA	80	-	-	-	-	376
		With S9
DMSO	0	121	8	15	17	9
Dinotefuran	1.2	106	10	18	22	9
	4.9	119	10	15	19	8
	20	121	10	14	17	8
	78	120	10	12	19	5
	313	116	7	18	13	7
	1250	118	10	13	14	6
	5000	112	10	11	14	8
2-AA	1	1006	-	-	-	-
	2	-	208	-	-	-
	10	-	-	845	-	-
	0.5	-	-	-	286	-
	2	-	-	-	-	71

 

Table 3: Summary of the incidence of revertant colonies for the main assay

Treatment	Dose	Mean (n = 3) ± SD revertant colonies/plate for strain:
	(µg/plate)	TA100	TA1535	WP2uvrA	TA98	TA1537
		Without S9
DMSO	0	101	5	25	12	3
Dinotefuran	313	111	7	26	12	3
	625	104	7	18	7	3
	1250	121	9	22	11	2
	2500	103	6	19	9	5
	5000	103	9	18	16	5
AF-2	0.01	727	-	-	-	-
NaN3	0.5	-	244	-	-	-
AF-2	0.01	-	-	106	-	-
AF-2	0.1	-	-	-	460	-
9-AA	80	-	-	-	-	578
		With S9
DMSO	0	104	9	24	18	12
Dinotefuran	313	102	8	24	21	8
	625	107	10	25	16	8
	1250	111	9	27	26	10
	2500	112	7	28	25	13
	5000	106	7	25	19	13
2-AA	1	665	-	-	-	-
	2	-	210	-	-	-
	10	-	-	799	-	-
	0.5	-	-	-	209	-
	2	-	-	-	-	81

Conclusions:

Interpretation of results:
negative

No appreciable cytotoxicity was observed in all strains, with and without metabolic activation at up to 5000µg/plate, the highest concentration evaluated. None of the strains showed an appreciable increase in the reversion frequency at any of the dose levels tested.

In the main assay, dinotefuran did not influence the growth of any strain tested at dose levels of up to 5000µg/plate. No appreciable increase in the reversion frequencies occurred in any strain tested in the dose range 313 - 5000µg/plate. In contrast, the positive control substances produced marked increases in the number of revertant colonies in all strains tested.

Under the conditions of this study, dinotefuran and/or metabolites does not induce gene mutations in the strains of S. typhimurium and E. coli used in the study at doses up to 5000µg/plate.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

2019 in vivo MN study additionally covered the endpoint aneugenicity and was negative.