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

Endpoint summary

Description of key information

For the oral (dietary) route, the main toxic effects reported in all species tested (rats, mice

and dogs) are reduced bodyweight gain and food consumption for subacute, subchronic and

chronic exposures. The only evidence of target organ toxicity was the observation of

increased cytoplasmic vacuolation of the adrenal cortex in a subchronic study in rats,

although the adversity of this finding was considered as being questionable. The lowest oral

dietary NOAEL is 22 mg/kg/day, observed in a subchronic (1 year) study in dogs.

For short-term oral gavage administration, investigated in developmental toxicity studies in

rats and rabbits, reduced bodyweight gain and food consumption also occurred in both

species. However, in rabbits these changes were accompanied by clinical signs

(hypoactivity, prone position, panting, flushed nose and ears, and tremors in one study) and

in one study by macroscopic pathology changes in the liver (pale discolouration) and

stomach (gray-white plaque in fundus, thickened gastric mucosa), although the toxicological

significance of the macroscopic changes is uncertain. The lowest short-term oral gavage

NOAEL is 52 mg/kg/day, observed in rabbits.

By the dermal route, dinotefuran does not cause systemic or local toxicity on repeated

subacute exposure.

By the inhalation route, a no-observed-adverse-effect-level for respirable dinotefuran was established in both sexes as 2.08mg/L, the maximum technically achievable aerosol concentration with a MMAD ± GSD of 1.55 ± 2.96µm.

Classification for repeated dose toxicity is not appropriate because severe, irreversible,

toxicity was not present at the guidance exposure levels given in the classification criteria of

Directive 67/548/EEC and Regulation (EC) 1272/2008.

Key value for chemical safety assessment

Toxic effect type:

concentration-driven

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28/10/1996 - 31/12/1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP, Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

(1981)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 82-1 (90-Day Oral Toxicity)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF 59 NohSan no. 4200 (1985)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

mouse

Strain:

other: Crl:CD1® (ICR)BR VAF/Plus ®

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: About 7 weeks old
- Weight at study initiation: 25.9 – 35.9 g for males. 18.8 – 32.2 g for females

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet: Diets were prepared approximately every 4 weeks [on Days -5 for Weeks 1-4], [on Day 23 for Weeks 5-8], [on Day 52 for Weeks 9-12] and [on Day 80 for Weeks 13 and 14].
- Mixing appropriate amounts with: Basal diet: Certified Rodent Diet #5002 meal
- Stability and homogeneity of the preparation: Stability of representative test diets containing 5000 or 50000ppm dinotefuran, bracketing the range of concentrations employed, was determined for 40 days at room temperature before start of the study. Stability analyses of diets containing 5000 or 50000ppm revealed dinotefuran to be stable for 40 days at room temperature. Further stability analyses on diets containing 500ppm demonstrated stability at room temperature for 40 days, at which time 100% of the initial concentration remained. The homogeneity analyses of multiple diet samples containing 500 or 50000ppm were within the ranges 101 - 106% and 96.0 - 97.4%, respectively, of theoretical concentrations.
- Achieved concentrations: Periodic analyses of all diets for achieved concentration showed dinotefuran to be present in the formulations at 91.6 to 113% nominal concentrations.
- Mean achieved dose levels: 0, 81, 844, 4442 and 10635mg/kg b.w./day (males) and 0, 102, 1064, 5414 and 11560mg/kg bw/day (females).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses for the concentration of dinotefuran in the dose preparations were conducted by Covance Laboratories Inc. using an analytical method, MP-MT25-MA, validated by Covance Laboratories Inc.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Daily, except where mice were fasted

Remarks:

Doses / Concentrations:
500 ppm
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
5000 ppm
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
25000 ppm
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
50000 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

10 mice per sex per dose

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: Based on preliminary results of a previous 4-week dietary study with dinotefuran (7.5.1. Weiler (1997)/4 week diet mouse/key). Slightly lower body weights for animals given diets containing 50000 ppm dinotefuran and minimal increases in total protein and albumin in males given diets containing 50000 ppm dinotefuran in a 13-week study. It was anticipated that the low-dose level would be the no-observed-effect-level. The mid- and mid-high-dose levels were selected as fractions of the high-dose level that were expected to result in dose-related effects.
- Post exposure observation period: none

Positive control:

None

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Morbidity/mortality checks were performed twice daily and a detailed clinical examination performed weekly.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded weekly throughout the study.

FOOD CONSUMPTION:
- Time schedule for examinations: Food consumption was recorded weekly throughout the study.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmic examinations were conducted pre-test and in Week 14.
- Dose groups that were examined: All mice

HAEMATOLOGY: Yes / No / No data
- Time schedule for collection of blood: Hematology was performed in Week 14. Blood samples were withdrawn after a period of at least 4 hours food deprivation.
- Anaesthetic used for blood collection: Yes, sodium pentobarbital
- Animals fasted: Yes, for at least 4 hours prior to sampling
- How many animals: 5 mice/sex/group

CLINICAL CHEMISTRY: Yes / No / No data
- Time schedule for collection of blood: Hematology was performed in Week 14. Blood samples were withdrawn after a period of at least 4 hours food deprivation.
- Anaesthetic used for blood collection: Yes, sodium pentobarbital
- Animals fasted: Yes, for at least 4 hours prior to sampling
- How many animals: 5 mice/sex/group

URINALYSIS: Yes / No / No data
- Time schedule for collection of urine: Week 14
- Animals fasted: Yes, for at least 4 hours prior to sampling
- How many animals: All mice

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

Where appropriate, data were analysed statistically at the 5% level by one-way analysis of variance on homogeneous or transformed data followed by Dunnett’s t-test where ANOVA proved significant. Levene’s test was used to evaluate the homogeneity of variance.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

See Table 1

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

See Table 2

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

See Table 2

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

There were no deaths and no treatment-related clinical signs at any dose level during the study, but both sexes at 50000ppm lost weight during the first week of treatment and subsequently showed a treatment-related depression of body weight gain (Table 1). The overall mean weight gains of both sexes were significantly (p < 0.05) lower than control values and at termination the group mean body weights were 15.4 and 21.9% lower than the controls in males and females, respectively. The overall weight gains of males at 25000ppm and females at 500, 5000 and 25000ppm were 15.9 to 31.4% lower than, but not significantly (p > 0.05) different from, the controls. Increased food spillage occurred during the first week in the groups treated at 25000 or 50000ppm and continued throughout the study at 50000ppm. Spillage is considered to indicate reduced diet palatability at concentrations ≥ 25000ppm and any apparent increase in food consumption is considered to represent spillage. There was no evidence of an effect on food consumption or food efficiency at dose levels up to 5000ppm.

There were no treatment-related ophthalmological and hematological effects at any dose level. All group mean hematological values were similar to, and not significantly (p > 0.05) different from, the control values. Treatment-related effects on serum chemistry after 13 weeks of treatment were confined to the group treated at 50000ppm (Table 2). The serum albumin concentration of males was slightly, but significantly (p < 0.05) higher than the control value by 17.2%. Urine pH was slightly lower in both sexes, but significantly different from the controls in the female group only. These minor differences from the controls were not associated with overt histopathological alterations and are considered not to be adverse effects. With the exception of serum urea concentration in females at 25000ppm which was significantly (p < 0.05) higher than the control value, all other serum and urine clinical chemistry values were not significantly (p > 0.05) different from the control values at all dose levels.

There were no primary treatment-related effects on absolute and relative organ weights at any dose level. The absolute weights of the heart and liver in females at 50000ppm and of the kidneys in both sexes were significantly (p < 0.05) lower than control values. The differences are considered to be a consequence of growth retardation since the organ/body weight ratios were not affected. A significant (p < 0.05) increase in the brain/body weight ratios in both sexes at 50000ppm is also considered to reflect growth retardation. There were no treatment-related macroscopic findings at necropsy at any dose level. There were no treatment-related histopathological alterations and the nature, severity and incidence of microscopic findings were similar in all treated and control groups.

Dose descriptor:

NOEL

Effect level:

25 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Dose descriptor:

NOAEL

Effect level:

25 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

Table 1: Summary of bodyweights, weight gains and food consumption

Interval	Males treated at (ppm):					Females treated at (ppm):
	0	500	5000	25000	50000	0	500	5000	25000	50000
	Group mean body weight (g)
Week 1	31.0	31.6	30.2	30.1	31.1	26.0	26.1	25.3	26.1	25.6
Week 2	32.7	33.2	32.0	31.2	30.2	27.3	27.2	26.5	26.8	24.0
Week 3	34.4	35.0	33.7	32.9	30.4*	28.9	28.5	27.6	28.0	24.7*
Week 4	34.6	34.9	34.2	32.8	30.9*	29.1	28.5	27.8	28.1	25.1*
Week 8	37.9	38.1	37.0	36.1	33.2*	31.5	30.7	30.0	30.2	26.6*
Week 14	41.7	41.9	40.3	39.1	35.3*	36.5	34.1	32.5*	33.3	28.5*
Overall gain	10.7	10.3	10.1	9.0	4.2*	10.5	8.0	7.2	7.2	2.9*
	Group mean food consumption (g/week)**
Weeks 1 - 4	45.1	43.6	43.6	43.7	49.6	41.4	43.2	43.6	44.5	46.1
Weeks 5 - 8	45.6	44.4	45.1	45.8	51.4	45.0	46.1	45.1	45.9	42.3
Weeks 9 - 13	40.5	40.4	40.2	41.7	45.3	42.6	42.1	42.6	43.7	41.9

* p < 0.05

** food consumption of animals not showing substantial food spillage

Table 2: Treatment related serum and urine clinical chemistry findings - Week 14

Sex	Dose level (ppm)	Mean serum albumin concentration ±SD (g/dL)	Mean urine pH (range)
Male	0	2.9±0.14	7.3 (6.5 - 8.5)
	500	3.1±0.31	7.5 (7.0 - 8.0)
	5000	3.2±2.1	7.5 (7.0 - 8.0)
	25000	3.3±0.18	6.9 (7.0 - 8.5)
	50000	3.4±0.26*	6.3 (6.0 - 8.0)
Female	0	3.4±0.09	7.2 (6.5 - 8.0)
	500	3.4±0.25	7.2 (6.5 - 7.5)
	5000	3.4±0.11	7.2 (6.5 - 8.0)
	25000	3.5±0.38	6.8 (6.5 - 7.5)
	50000	3.6±0.29	6.6* (6.0 - 7.0)

* p < 0.05

Conclusions:

No target organs were identified in either sex at the highest dose level employed. The no-observed-effect-level (NOEL) and no-observed-adverse-effect-level (NOAEL) were established as 25000ppm in both sexes, equivalent to dose levels of 4442mg/kg bw/day (males) and 5414mg/kg bw/day (females), based on the occurrence of reduced weight gain in both sexes at 50000ppm. In addition, non-adverse effects of increased serum albumin concentration in males and reduced urine pH in both sexes occurred at 50000ppm.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28/10/1996 - 31/12/1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP, Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

(1981)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 82-1 (90-Day Oral Toxicity)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF 59 NohSan no. 4200 (1985)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:CD® (SD)BR VAF/Plus ®

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: About 7 weeks old
- Weight at study initiation: 235-284 g for males. 165-228 g for females

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet: Diets were prepared approximately every 4 weeks [on Days -5 for Weeks 1-4], [on Day 23 for Weeks 5-8], [on Day 52 for Weeks 9-12] and [on Day 80 for Weeks 13 and 14].
- Mixing appropriate amounts with: Basal diet: Certified Rodent Diet #5002 meal
- Stability and homogeneity of the preparation: Stability of representative test diets containing 5000 or 50000ppm dinotefuran, bracketing the range of concentrations employed, was determined for 40 days at room temperature before start of the study. Stability analyses performed at 5000 and 50000ppm (unpublished report no. CHW 6648-125, 7.5.1. Weiler (1997)/4 week diet rat/key) and at 500ppm revealed dinotefuran to be stable at room temperature for 40 days, at which time 101% nominal concentration remained. The homogeneity analyses of multiple diet samples containing 500 or 50000ppm were within the ranges 101 - 106% and 96.0 - 97.4%, respectively, of theoretical concentrations.
- Achieved concentrations: Periodic analyses for achieved concentration showed dinotefuran to be present in the formulations at 91.6 to 113% nominal concentrations.
- Mean achieved dose levels: 0, 500, 5000, 25000 and 50000 ppm in the diet (mean achieved dose levels 0, 34, 336, 1623 and 3156 mg/kg bw/day in males and 0, 38, 384, 1871 and 3616 mg/kg bw/day in females.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses for the concentration of dinotefuran in the dose preparation were conducted by Corning Hazleton Inc. using an analytical method, MP-MT25-MA, validated by Corning Hazleton Inc.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Daily, except where animals were fasted

Remarks:

Doses / Concentrations:
500 ppm
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
5000 ppm
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
25000 ppm
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
50000 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

10 rats per sex per dose

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: Based on the results of a 4-week dietary study in which NOAEL was established as 50000 ppm (equivalent to 3720 and 4222 mg/kg/day, in males and females, respectively)
- Post exposure observation period: none

Positive control:

None

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Morbidity/mortality checks were performed twice daily and a detailed clinical examination performed weekly.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded weekly throughout the study.

FOOD CONSUMPTION:
- Food consumption was recorded weekly throughout the study.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmic examinations were conducted pre-test and in week 14.
- Dose groups that were examined: All rats

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Pre-test and during Week 14
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes, overnight
- How many animals: All rats

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Pre-test and during Week 14
- Animals fasted: Yes, overnight
- How many animals: All rats

URINALYSIS: Yes
- Time schedule for collection of urine: Pre-test and during Week 14
- Animals fasted: Yes, overnight

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

Where appropriate, data were analysed statistically at the 5% level by one-way ANOVA on homogeneous or transformed data followed by Dunnett’s multiple comparison t-test where ANOVA was significant. Levene’s test was used to evaluate the homogeneity of variance.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

See Table 1

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

See Table 1

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

See Table 2

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

See Table 2

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See Table 3

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See Table 3

Details on results:

There were no deaths and no treatment-related clinical signs during the study. Males and females treated at 50000ppm lost weight during week 1, but subsequently gained weight. The animals of both sexes treated at 25000 or 50000ppm, and females treated at 5000ppm, showed statistically significant (p < 0.05) and dose-related reductions in overall body weight gain (Table 1). At termination, the mean body weights of these groups were 7.2 to 24.1% lower than control values (Table 1). The mean weekly food consumption of both sexes at 25000 or 50000ppm was significantly (p < 0.05) reduced for at least 11 weeks of the treatment period, and the overall mean food consumption was 11.5 to 24.5% lower than control values. Although the overall weight gain of females at 5000ppm was significantly lower than the control value and overall food consumption was 8.7% lower than the control value (p < 0.05 on 6 occasions), the observations are considered not to be adverse effects because the group mean body weight and food consumption remained within 10% of the control values throughout the study.

There were no treatment-related ocular lesions at any dose level. Treatment-related effects on hematology and serum clinical chemistry after 13 weeks of treatment were confined to the group treated at 50000ppm (Table 2). Activated partial thromboplastin times (APTT) were slightly shorter than control values and urea nitrogen concentrations were slightly elevated in both sexes, but were significantly (p < 0.05) different from the controls in males only. Males at 50000ppm also showed slightly, but significantly (p < 0.05) lower blood glucose (Glu), total protein (Prot) and globulin (Glob) concentrations. These minor differences from the controls were not associated with overt histopathological alterations and are considered not to be of toxicological significance or adverse effects. All other hematological and serum clinical chemistry parameters were comparable to control group values. Urinalysis profiles were unaffected by treatment at all dose levels.

There were no treatment-related macroscopic findings at necropsy. There were no effects on absolute organ weights or ratios that are considered to be a direct effect of treatment at any dose level. However, absolute heart, kidney, liver and spleen weights were significantly (p < 0.05) lower and their ratios relative to body weight and/or brain weight were significantly different from the controls in the groups treated at 25000 and 50000ppm. The absolute weights of the adrenals and pituitary were lower in females and the relative weights of brain and testes were higher in animals at 50000ppm. Since the body weights of these groups were significantly reduced at termination and because there were no correlating histopathological changes in these organs, the differences from control values are considered to be incidental to treatment or secondary to substantially reduced weight gain. Treatment-related histopathological alterations were confined to increased cytoplasmic vacuolation of the adrenal cortex in both sexes treated at 25000 and 50000ppm and in males at 5000ppm (Table 3). The vacuolation was apparent in both the zona glomerulosa and zona fasciculata of the males but was confined to the zona glomerulosa in the females. The severity of the lesion was graded as minimal or slight in all instances except for one female at 50000ppm that was graded moderate. Minimal to moderate adrenal cortical vacuolation is considered not to be an adverse finding in this study since there were no correlating clinical pathology findings indicating functional deficit. The nature, incidence and severity of all other histopathological findings in animals treated at 0 or 50000ppm did not indicate an effect of treatment with dinotefuran.

Dose descriptor:

NOEL

Effect level:

500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Dose descriptor:

NOAEL

Effect level:

25 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

Tabe 1: Summary of bodyweight gain and food consumption

Sex	Dose level (ppm)	Overall group mean weight gain (g)	Group mean terminal body weight (g)	Overall mean food consumption (g/wk)a
Male	0	315	572	208
	500	316	568	204
	5000	295	552	201
	25000	257*	515*	184
	50000	190*	446*	157
Female	0	141	345	160
	500	131	326	146
	5000	118*	320*	146
	25000	96*	291*	131
	50000	67*	262*	121

* p < 0.05; a statistical analysis not performed on overall group mean data

Table 2: Treatment related effects on hematology and serum clinical chemistry - Week 14

Sex	Dose level	Group mean hematology and serum chemistry values:
	(ppm)	APTT (sec)	UN (mg/dL)	Glu (mg/dL)	Prot (g/dL)	Glob (g/dL)
Male	0	14.4	14	102	7.9	3.1
	500	13.8	13	111	7.7	2.9
	5000	14.8	14	104	7.8	2.9
	25000	13.7	15	98	7.6	2.8
	50000	13.1*	17*	85*	7.5*	2.7*
Female	0	13.2	16	102	8.3	2.6
	500	12.6	16	107	8.3	2.7
	5000	12.8	15	105	8.2	2.5
	25000	12.6	16	98	8.1	2.6
	50000	12.2	19	104	8.0	2.7

* p < 0.05

Table 3: Treatment related histopathological alterations in the adrenal cortex

Sex	Dose level (ppm)	Incidence (%) of increased vacuolation in zona glomerulosa	Incidence (%) of increased vacuolation in zona fasciculata
Male	0	0	10
	500	0	0
	5000	30	20
	25000	20	30
	50000	40	50
Female	0	0	0
	500	0	0
	5000	0	0
	25000	60	0
	50000	100	0

Conclusions:

No target organs were identified in either sex at the highest dose level employed. A no-observed-effect-level (NOEL) for all effects was established as 500ppm diet in both sexes, equivalent to a dose level of 34mg/kg bw/day (males) and 38mg/kg bw/day (females), based on the occurrence of marginally lower growth rate in females and adrenal cytoplasmic vacuolation in males at 5000ppm, adrenal cytoplasmic vacuolation in both sexes at 25000 and 50000ppm and increased serum urea and reduced serum glucose, total protein and globulin concentrations in males at 50000ppm.
A no-observed-adverse-effect-level (NOAEL) was established as 25000ppm in both sexes, equivalent to dose levels of 1623mg/kg bw/day (males) and 1871mg/kg bw/day (females), based on the occurrence of marked growth retardation and reduced food consumption at a concentration of 50000ppm, all other effects being considered not to be toxicologically relevant.

Reference 3

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25/07/1996 - 15/12/1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP, Guideline study: On day 3, one animal (Group 4 male) was found dead; necropsy findings suggested pre-existing renal disease that was unlikely to be related to the administration of the test material. Based on the findings, it was decided to replace this animal with one of the extra animals. Deviations from 92/69/EEC - none; special functional observations and motor activity assessment required by OECD 407 not performed.

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

(1995)

Deviations:

yes

Remarks:

special functional observations and motor activity assessment required by OECD 407 not performed

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

mouse

Strain:

other: Crl:CD1®(ICR)BR VAF/Plus®

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: About 7 weeks old
- Weight at study initiation: 18.5 – 36.2 g for males. 22.4 – 28.9 g for females

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet: Diets were prepared once before initiation of treatment
- Mixing appropriate amounts with: Basal diet: Certified Rodent Diet #5002 meal
- Stability and homogeneity of the preparation: Stability of representative test diets containing 5000 or 50000ppm dinotefuran, bracketing the range of concentrations employed, was determined for 40 days at room temperature before start of the study. Stability analyses revealed dinotefuran to be stable for 40 days at room temperature. Homogeneity analysis of multiple samples of diets containing 5000 or 50000ppm showed dinotefuran concentrations to be in the range 93.4 and 97.6% of theoretical concentrations.
- Achieved concentrations: Analysis of achieved concentrations demonstrated dinotefuran to be present in the formulations at 95.6 to 98.4% nominal concentrations.
- Mean achieved dose levels: 0, 901, 4612 and 10303mg/kg bw/day (males) and 0, 1043, 5359 and 12289mg/kg bw/day (females).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses for the concentration of dinotefuran in the dose preparations were conducted by Corning Hazleton Inc. using an analytical method, MP-MT25-MA, validated by Corning Hazleton Inc.

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

Daily, except when animals were fasted

Remarks:

Doses / Concentrations:
5000 ppm
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
25000 ppm
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
50000 ppm
Basis:
nominal in diet

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: Dose levels were selected by the Sponsor based on preliminary work.
- Post exposure observation period: none

Positive control:

None

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Morbidity/mortality checks were performed twice daily and a detailed clinical examination was performed weekly.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded weekly throughout the study.

FOOD CONSUMPTION:
- Time schedule for examinations: Food consumption was recorded weekly throughout the study.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: During Week 5
- Anaesthetic used for blood collection: Yes, using sodium pentobarbital
- Animals fasted: Yes, for at least 4 hours prior to blood collection
- How many animals: 5 mice/sex/group

CLINICAL CHEMISTRY: Yes / No / No data
- Time schedule for collection of blood: During Week 5
- Anaesthetic used for blood collection: Yes, using sodium pentobarbital
- Animals fasted: Yes, for at least 4 hours prior to blood collection
- How many animals: 5 mice/sex/group

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

Where appropriate, data were analysed statistically at the 5% level by one-way ANOVA on homogeneous or transformed data followed by Dunnett’s multiple comparison t-test where ANOVA proved significant. Levene’s test was used to evaluate the homogeneity of variance.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

See Table 1

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

See Table 1

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

See Table 2

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

One male treated at 50000ppm was found dead on day 3. Macroscopic pathology revealed multiple areas of discoloration in the renal cortex and medulla of both kidneys, mucoid material in the renal pelves and bilateral enlargement of the lumbar and renal lymph nodes. These findings indicated the probable cause of death to be pre-existing renal disease and unrelated to the administration of dinotefuran. The animal was replaced.
There were no other deaths and no treatment-related clinical signs at any dose level during the study. Both sexes treated at 25000 or 50000ppm showed a dose-related depression of overall body weight gain of between 46.2 and 85.5%. Males and females treated at 50000ppm lost weight during the first week of treatment (Table 1) but subsequently gained weight at a comparable rate to the controls. Marked food spillage by the groups treated at 25000 or 50000ppm suggested these test diets were less palatable than untreated diet, and precluded a valid assessment of food consumption for most time points. Therefore, initial weight loss and/or overall depressed weight gain at 25000 and 50000ppm is considered to be a reflection of reduced palatability of the diets. At 5000ppm, there was no evidence of an effect on food consumption.

There were no treatment-related effects on the hematological profile at any dose level. Treatment-related effects on serum chemistry were confined to the male group treated at 50000ppm and comprised slightly, but significantly (p < 0.05), higher total serum protein and albumin concentrations (Table 2). These minor differences from the controls were not associated with overt histopathological changes and are considered not to be adverse effects. All other serum chemistry values were unaffected by treatment at all dose levels.

There were no treatment-related effects on absolute and relative organ weights and no treatment-related macroscopic or microscopic histopathological alterations at any of the dose levels employed.

Dose descriptor:

NOEL

Effect level:

5 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Dose descriptor:

NOAEL

Effect level:

50 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Equivalent to dose levels of 10303mg/kg bw/day (males) and 12289mg/kg bw/day (females). Since reduced weight gain was a consequence of reduced diet palatability and the minor changes in serum chemistry are considered not to be adverse.

Critical effects observed:

not specified

Table 1: Treatment related effects on group mean bodyweight and overall weight gain

Week of study	Males treated at (ppm):				Females treated at (ppm):
	0	5000	25000	50000	0	5000	25000	50000
1	30.7	32.1	32.6	32.8	25.5	26.1	26.5	26.0
2	33.1	33.7	32.9	30.9	27.0	27.5	27.0	24.9*
3	34.9	35.3	34.0	32.0*	28.4	29.0	27.9	26.0*
4	35.4	35.7	34.1	33.3	28.9	29.0	28.3	27.0
5	36.2	37.0	35.4	33.6	29.4	30.4	28.6	27.8
Overall gain (g)	5.5	4.9	2.8	0.8	3.9	4.3	2.1	1.8

* p < 0.05

Table 2: Treatment realated serum clinical chemistry finding - Week 5

Sex	Dose level (ppm)	Mean serum total protein±SD (g/dL)	Mean serum albumin±SD (g/dL)
Male	0	4.7±0.23	3.2±0.13
	5000	4.8±0.26	3.2±0.30
	25000	5.0±0.25	3.3±0.18
	50000	5.1*±0.18	3.6*±0.23
Female	0	4.8±0.15	3.5±0.13
	5000	5.0±0.22	3.4±0.26
	25000	5.1±0.14	3.7±0.09
	50000	4.9±0.20	3.5±0.13

* p < 0.05

Conclusions:

No target organs were identified in either sex at the highest dose level employed. A no-observed-effect-level (NOEL) was established as 5000ppm diet in both sexes, equivalent to dose levels of 901mg/kg bw/day (males) and 1043mg/kg bw/day (females), based on the occurrence of reduced weight gain in both sexes at dose levels ≥25000ppm and slightly elevated total serum protein and albumin concentrations in males at 50000ppm. Since reduced weight gain was a consequence of reduced diet palatability and the minor changes in serum chemistry are considered not to be adverse, a no-observed-adverse-effect-level (NOAEL) was established in both sexes as 50000ppm, equivalent to dose levels of 10303mg/kg bw/day (males) and 12289mg/kg bw/day (females).

Reference 4

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25/07/1996 - 15/12/1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP, Guideline study: Deviations from 92/69/EEC - none; special functional observations and motor activity assessment required by OECD 407 not performed.

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

(1995)

Deviations:

yes

Remarks:

special functional observations and motor activity assessment required by OECD 407 not performed

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:CD® [SD]BR VAF/Plus ®

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: About 7 weeks old
- Weight at study initiation: 241-287 g for males. 137-176 g for females

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet: Diets were prepared once before initiation of treatment
- Mixing appropriate amounts with: Basal diet: Certified Rodent Diet #5002 meal
- Achieved concentration: The analyses for achieved concentration showed dinotefuran to be present in the formulations at 99.0 to 99.6% nominal concentrations.
- Stability and homogeneity of the preparation: Stability analyses revealed dinotefuran to be stable for 40 days at room temperature, at which time 99.4 and 98.4% of the initial concentrations remained in the 5000 and 50000ppm diets, respectively. Mean values of the homogeneity analyses in multiple diet samples containing 5000 or 50000ppm were in the ranges 98.4 - 99.0% and 98.0 - 101%, respectively, of theoretical concentrations.
- Mean achieved dose levels: 0, 390, 1814 and 3720mg/kg bw/day (males) and 0, 450, 2183 and 4222mg/kg bw/day (females).
- Post exposure period: none

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses for the concentration of dinotefuran in the dose preparations were conducted by Corning Hazleton Inc. using an analytical method, MP-MT25-MA, validated by Corning Hazleton Inc.

Duration of treatment / exposure:

4 weeks (except where animals were fasted).

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
5000 ppm
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
25000 ppm
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
50000 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

5 rats per sex per dose

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: Dose levels were selected by the Sponsor based on preliminary work.
- Post exposure observation period: none

Positive control:

None

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The animals were observed twice daily for viability. A detailed clinical examination was performed weekly.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded weekly throughout the study.

FOOD CONSUMPTION:
- Time schedule for examinations: Food consumption was recorded weekly throughout the study.

HAEMATOLOGY: Yes / No / No data
- Time schedule for collection of blood: During Week 5
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes, overnight
- How many animals: Each test rat

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: During Week 5
- Animals fasted: Yes, overnight
- How many animals: Each test rat

URINALYSIS: Yes
- Time schedule for collection of blood: During Week 5
- Animals fasted: Yes, overnight
- How many animals: Each test rat

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

Where appropriate, data were analysed statistically at the 5% level by one-way ANOVA on homogeneous or transformed data followed by Dunnett’s multiple comparison t-test where ANOVA proved significant. Levene’s test was used to evaluate the homogeneity of variance.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

See Table 1

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

See Table 1

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

See Table 2

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

no effects observed

Details on results:

There were no deaths and no treatment-related clinical signs at any dose level during the study. Females treated at 50000ppm lost weight during the first week of treatment, but subsequently gained weight. Males and females treated at 50000ppm and males at 25000ppm showed significant (p < 0.05) reductions in overall weight gain of 46.9, 21.5 and 49.3%, respectively (Table 1). Although the female group treated at 25000ppm showed a 25.4% reduction in overall weight gain, the difference from the controls was not statistically significant. Reduced weight gain in both sexes at 50000ppm and males at 25000ppm was associated with reduced overall food consumption of 10.4 to 17.6%. The food consumption of these groups was significantly lower than the controls during the first week of treatment and also during the second week in males at 50000ppm. Therefore, reduced weight gain is considered to be due to reduced diet palatability and not to be an adverse effect of dinotefuran. There were no treatment-related effects on weight gain and food consumption at 5000ppm.

There were no treatment-related effects on hematology and urinalysis parameters at any dose level. Treatment-related effects on serum clinical chemistry were confined to the male groups treated at 25000 and 50000ppm (Table 2). The group mean glucose concentration at 50000ppm was slightly, but significantly (p < 0.05) reduced, and the group mean cholesterol concentrations at 25000ppm and 50000ppm were significantly (p < 0.05) increased relative to the controls. However, these minor differences from the controls were not apparent in the females at any dose level and were not associated with overt histopathological alterations at 50000ppm. Therefore, they are considered not to be adverse effects but a manifestation of minor alterations in carbohydrate and lipid metabolism associated with reduced body weight gain. There were no other treatment-related effects on serum clinical chemistry parameters at any dose level.

There were no effects on organ weights and ratios considered to be directly related to treatment with dinotefuran. A number of absolute organ weights were slightly, but significantly (p < 0.05), lower in males at 50000ppm (spleen, heart, kidneys, liver) and females at 50000ppm (heart and ovaries). However, these differences were not apparent in the corresponding body weight ratios. Therefore, the differences in absolute organ weights are considered to be a consequence of the lower terminal body weights (Table 2). There were no treatment-related macroscopic lesions at any dose level and no treatment-related microscopic findings at 50000ppm. The nature and incidences of all histopathological alterations were comparable in the control and treated groups.

Dose descriptor:

NOEL

Effect level:

5 000 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: see 'Remark'

Dose descriptor:

NOEL

Effect level:

25 000 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: see 'Remark'

Dose descriptor:

NOAEL

Effect level:

50 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Equivalent to a dose level of 3720mg/kg bw/day (males) and 4222mg/kg bw/day (females). Based on the absence of toxicologically significant effects at 50000ppm.

Critical effects observed:

not specified

Table 1: Summary of bodyweight gain and food consumption

Sex	Dose level (ppm)	Overall group mean weight gain (g)	Group mean terminal body weight (g)	Overall mean food consumption (g/wk)a
Male	0	177	404.0	193
	5000	173	403.1	193
	25000	139*	378.6	173
	50000	94*	335.5*	159
Female	0	67	199.2	119
	5000	53	197.6	120
	25000	50	188.2	111
	50000	34*	172.6	99

* p < 0.05; a statistical analysis not performed on overall group mean data

Table 2: Treatment related effects on serum clinical chemistry - Week 5

Sex	Dose level (ppm)	Group mean serum glucose concentration (mg/dL)	Group mean serum cholesterol concentration (mg/dL)
Male	0	118	56
	5000	119	52
	25000	111	70*
	50000	101*	80*
Female	0	105	85
	5000	105	70
	25000	103	83
	50000	100	86

* p < 0.05

Conclusions:

No target organs were identified in either sex at the highest dose level employed. The no-observed-effect-level (NOEL) for all effects was established as 5000ppm diet in males and 25000ppm in females, equivalent to dose levels of 390mg/kg bw/day (males) and 2183mg/kg bw/day (females), based on the occurrence of reduced weight gain and increased serum cholesterol in males at 25000ppm, reduced weight gain in females at 50000ppm and reduced serum glucose concentration in males at 50000ppm.
A no-observed-adverse-effect-level (NOAEL) was established as 50000ppm, equivalent to a dose level of 3720mg/kg bw/day (males) and 4222mg/kg bw/day (females), based on the absence of toxicologically significant effects at 50000ppm.

Reference 5

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27/10/1997 - 13/05/1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP, Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)

Version / remarks:

(1981)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 82-1 (90-Day Oral Toxicity)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF 59 NohSan no. 4200 (1985)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 5-6 months old
- Weight at study initiation: 7.7 – 8.8 kg for males. 6.3 – 8.8 kg for females

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet: Approximately weekly for the first 5 weeks and approximately every other week thereafter or as needed due to changes in dose levels.
- Mixing appropriate amounts with: Basal diet: Certified canine meal #8727 CM
- Stability and homogeneity of the preparation: Stability of representative test diets containing 5000 or 50000ppm dinotefuran, bracketing the range of concentrations employed, was determined for 40 days at room temperature before start of the study. Stability analyses of diets containing 5000 or 50000ppm revealed dinotefuran to be stable for 40 days at room temperature. Further stabilitThe homogeneity analyses of multiple diet samples containing 500 or 50000ppm were within the ranges 101 - 106% and 96.0 - 97.4%, respectively, of theoretical concentrations.y analyses on diets containing 500ppm demonstrated stability at room temperature for 40 days, at which time 100% of the initial concentration remained.
- Achieved concentrations: Periodic analyses of all diets for achieved concentration showed dinotefuran to be present in the formulations at 91.6 to 113% nominal concentrations.
- Mean achieved dose levels: 0, 58, 307 and 862mg/kg bw/day (males) and 0, 58, 323 and 950mg/kg bw/day (females).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses for the concentration of dinotefuran in the dose preparations were conducted by Covance Laboratories Inc. using an analytical method, MP-MT28_MA, validated by Covance Laboratories Inc.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Dose preparations were offered for an approximate 4-hour feeding period per day for the first 2 days of treatment and ad libitum, 7 days per week, beginning on Day 3 for at least 13 weeks, except when dogs were fasted.

Remarks:

Doses / Concentrations:
1600 ppm
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
8000 ppm
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
40000/24000 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

4 dogs per sex per dose

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: Based on results of previous short term dietary studies with dinotefuran. Diets containing 50000 ppm dinotefuran were not well tolerated, but diets containing 40000 ppm dinotefuran were tolerated for at least 1 week. Based on the results of this study, it was anticipated that the dogs would tolerate the high-dose diet of 40000 ppm. It was also anticipated that the low-dose level would be the no-observed-effect-level. The mid-dose level was expected to result in dose-related effects.
- Post exposure observation period: none

Positive control:

None

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Morbidity/mortality checks were performed twice daily and clinical signs were recorded daily.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded weekly and at necropsy.

FOOD CONSUMPTION:
- Time schedule for examinations: Food consumption was measured daily for one week pre-dose and for the first 2 weeks of treatment and weekly thereafter.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water consumption was recorded for 2 days/week throughout the treatment period.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Pre-test and during Week 14
- Dose groups that were examined: All dogs

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Pre-test and in Weeks 5 and 14
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes, overnight
- How many animals: All dogs

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Pre-test and in Weeks 5 and 14
- Animals fasted: Yes, overnight
- How many animals: All dogs

URINALYSIS: Yes
- Time schedule for collection of urine: Pre-test and in Weeks 5 and 14
- Animals fasted: Yes, overnight

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

Where appropriate, data were analysed statistically at the 5% level by one-way ANOVA on homogeneous or transformed data followed by Dunnett’s multiple comparison t-test where ANOVA proved significant. One-way ANCOVA was used to analyse body weights using initial body weight as covariate.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

See "Details on results"

Mortality:

mortality observed, treatment-related

Description (incidence):

See "Details on results"

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

See Table 1

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

See Table 2

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

See "Details on results"

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

See Table 3

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

See Table 3

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

There were no deaths during the study but the highest diet concentration was reduced to 30000ppm on day 5 and further reduced to 24000ppm on day 12 due to very low food consumption. Markedly lower food consumption at  30000ppm is considered to reflect diet unpalatability. Treatment-related clinical signs were confined to animals treated at 30000/40000ppm. Discolored feces, few or no feces, thinness, slightly reduced activity and pale gums occurred in some animals. Two males and one female initially treated at 40000ppm showed black feces for one or 2 days, but the occurrence may be related to stress resulting from offering unpalatable diet, rather than a primary effect of dinotefuran. One male treated at 40000/30000ppm showing liquid/mucoid feces was diagnosed with enteritis on day 8 and was taken off dose for 4 days. There were no other clinical signs after reduction of the dietary concentration to 24000ppm. Body weights (Table 1) were significantly reduced in both sexes at 24000ppm from week 2 resulting in overall reductions in weight gain of 33.3 and 31.4% in males and females, respectively. However, a large proportion of the reduced weight gain was associated with the administration of 30000 - 40000ppm during the first 12 days of treatment, and subsequent treatment at 24000ppm resulted in only marginally lower weight gains. Females at 1600 and 8000ppm also showed significantly lower body weights during the last 8 weeks of treatment and lower overall weight gains (34.3 and 31.4%, respectively) but these are considered not to be adverse effects since there were no adverse clinical signs or pathological findings.

Food consumption was markedly and significantly (p < 0.05) reduced in both sexes treated at 30000/40000ppm, indicating reduced diet palatability. Although food consumption increased after the reduction of the diet concentration to 24000ppm, reduced food consumption persisted in both sexes throughout the study (Table 2). There was a concomitant decrease in water consumption during the first 11 days of treatment at concentrations of  30000/40000ppm. Subsequently, the water consumption of the males remained depressed, but female water consumption was comparable to, or higher than, the control females as from day 24. The food consumption of females treated at 1600 and 8000ppm was also low in comparison with the female control consumption. However, the differences in food consumption are considered not to be toxicologically relevant because they were not dose-related and the pre-dose food consumption at 1600ppm was 16.4% lower than the control consumption. Water consumption of both sexes at 1600 or 8000 ppm was not affected by treatment.

There were no ocular lesions in any animal after 13 weeks of treatment and there were no treatment-related hematological changes in either sex at any dose level. Males and females at 24000ppm showed slightly but significantly (p < 0.05) reduced serum ALT activity relative to both pre-dose values and the controls after 5 and 13 weeks treatment (Table 3). Urine pH of males at 24000ppm was marginally lower than control values in weeks 5 and 14. These findings are considered not be adverse effects and of no toxicological relevance in the absence of correlating histopathological alterations. There were no other treatment-related changes in the hematology, serum chemistry and urinalysis profiles at any dose level.

All organ weights and ratios were unaffected by treatment at all dose levels and none at 24000ppm was significantly different from the controls (p > 0.05). There were no treatment-related macroscopic or microscopic histopathological alterations at any of the dose levels employed. Although hemorrhage in the mesenteric and/or mandibular lymph nodes occurred in 3 of the 4 males treated at 24000ppm, its occurrence is considered incidental to treatment with dinotefuran since hemorrhage was not evident in other organs and none of the macroscopic or clinical pathology observations suggested a hemorrhagic condition in these animals. All other histopathological alterations occurred sporadically and without regard to sex or dose level.

Dose descriptor:

NOEL

Effect level:

8 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Dose descriptor:

NOAEL

Effect level:

8 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

Table 1: Selected group mean bodyweight data

Sex / dose level (ppm)	Mean body weight (kg) at week:
	1	2	3	4	8	14
Males:
0	8.1	8.9	9.5	9.9	10.8	11.7
1600	8.0	9.1	9.4	9.9	11.0	11.8
8000	8.2	8.9	9.2	9.6	10.6	11.5
24000	8.3	7.9*	8.6*	9.1*	10.0*	10.7*
Females:
0	7.1	7.9	8.3	8.7	9.8	10.6
1600	7.0	7.7	8.0	8.2*	8.8*	9.3*
8000	7.2	7.8	8.2	8.4	9.0*	9.6*
24000	7.0	6.8*	7.3*	7.8*	8.6*	9.4*

* p < 0.05

Table 2: Representative group mean food and water consumption data

Sex	Dose level (ppm)	Mean food consumption (g/week) in week:
		1	2	4	8	13
Male	0	2951	2919	2875	2958	2990
	1600	2913	2671	2645	2528	2722
	8000	2555	2603	2686	2708	2896
	24000	1394*	2381	2570	2391*	2411*
Female	0	2712	2863	2773	2722	2657
	1600	2406	2311*	2212*	2203*	2102*
	8000	2588	2557	2392*	2350	2631
	24000	869*	2240*	2295*	2207*	2402
		Mean water consumption (g/week) on day:
		2	10	24	52	86
Male	0	915	1455	1423	1550	1505
	1600	780	958*	1090	980*	1095
	8000	743	1260	1295	1270	1313
	24000	138*	798*	1165	963*	1000*
Female	0	573	1030	945	977	980
	1600	503	903	790	658	833
	8000	390	818	735	683	747
	24000	38*	748	1035	940	1103

* p < 0.05

Table 3: Treatment related serum clinical chemistry findings

Sex	Mean serum ALT activity (IU/L)±SD in:			Mean urine pH (range) in:
dose level (ppm)	Week -1	Week 5	Week 14	Week -1	Week 5	Week 14
Males:
0	25±2.9	27±3.3	32±1.0	7.0 (7.0 - 7.0)	8.1 (7.5-8.5)	7.6 (7.0-8.5)
1600	30±7.3	24±0.5	27±1.9	6.9 (6.5 - 7.5)	7.5 (6.5-8.5)	7.6 (7.5-8.0)
8000	28±5.1	22±6.8	27±4.8	6.9 (6.5 - 7.0)	6.8 (6.0-7.5)	7.8 (7.0-8.5)
24000	29±5.5	18*±1.6	19*±4.3	7.0 (6.5 - 7.5)	6.5 (6.0-7.0)	6.8 (6.0-7.5)
Females:
0	32±7.7	26±3.5	29±6.7	6.8 (6.5 - 7.5)	6.9 (6.5-7.0)	6.6 (6.5-7.0)
1600	28±4.8	27±3.8	26±3.6	7.1 (6.0 - 7.5)	6.8 (6.5-7.0)	7.3 (6.5-8.0)
8000	26±5.9	24±5.4	22±4.4	7.1 (7.0 - 7.5)	6.5 (6.0-7.0)	7.1 (7.0-7.5)
24000	25±2.6	14*±1.5	20*±2.1	6.6 (6.5 - 7.0)	6.5 (6.0-7.0)	6.6 (6.0-7.0)

* p < 0.05

Conclusions:

No target organs were identified in either sex at the highest dose level employed. A no-observed-effect-level (NOEL) and NOAEL were established as 8000ppm in both sexes, equivalent to dose levels of 307mg/kg bw/day (males) and 323mg/kg bw/day (females), based on the occurrence of reduced food consumption, body weight gain and serum ALT activity in both sexes, and marginally reduced urine pH in males at 24000 - 40000ppm.
The reviewer considers the established NOAEL to be a conservative value because reduced body weight gain was not apparent in either sex during the 11 weeks of treatment at 24000ppm.

Reference 6

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20/05/1998 - 10/12/1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP, Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 452 (Chronic Toxicity Studies)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 83-1 (Chronic Toxicity)

Version / remarks:

(1982)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF 59 NohSan 4200 (1985)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 5-5.5 months old
- Weight at study initiation: 8.0 – 9.8 kg for males. 7.1 – 8.9 kg for females

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet: Diets were prepared at least monthly
- Mixing appropriate amounts with: Basal diet: Certified canine meal #8727 CM
- Stability and homogeneity of the preparation: The homogeneity analyses of multiple diet samples containing 640 or 16000ppm were within the ranges 96.7 - 116% and 99.4 - 101%, respectively, of theoretical concentrations. To evaluate the stability of representative test diets containing 100, 640 or 30000ppm dinotefuran was taken and analysed before initiation of treatment and after storage of a refrigerator for 29 days, then at room temperature for 8 or 9 days except one set from the 640 and 30000ppm diets only was stored in a refrigetor for 15 days, then at room temperature for 8 or 9 days. Stability analyses of diets containing 100, 640 or 30000ppm revealed dinotefuran to be stable for 29 days under refrigerated conditions followrd by 8 or 9 days at room temperature.
- Achieved concentrations: Periodic analyses of all diets for achieved concentration showed dinotefuran to be present in the formulations at 88.8 to 129% of the theoretical concentrations.
- Mean achieved dose levels: 0, 20, 111 and 559mg/kg bw/day (males) and 0, 22, 108 and 512mg/kg bw/day (females).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses for the concentration of dinotefuran in the dose preparations were conducted by Covance Laboratories Inc. using an analytical methodm MP-MT28-MA, validated by Covance Laboratories Inc.

Duration of treatment / exposure:

52 weeks

Frequency of treatment:

Daily, except when dogs were fasted

Remarks:

Doses / Concentrations:
640 ppm
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
3200 ppm
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
16000 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

4 dogs per sex per dose

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: Based on results of a previous 13-week dietary study with dinotefuran (study report number 6648-128; 7.5.1. Weiler (1999)/13 week diet dog/key). Based on the results of this study, it was anticipated that the dogs would tolerate the high-dose diet of 16000 ppm. It was anticipated that the low-dose level would be the no-oberserved-effect-level. The mid-dose level was expected to result in dose-related effects.
- Post exposure observation period: none

Positive control:

None

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The animals were observed twice daily for morbidity / mortality and daily for clinical signs of poor health or abnormal behavior.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded weekly for 16 weeks and at 4-week intervals thereafter.

FOOD CONSUMPTION:
- Time schedule for examinations: Food consumption was recorded weekly for 16 weeks and at 4-week intervals thereafter.

WATER CONSUMPTION : Yes
- Time schedule for examinations: Water consumption was recorded weekly for 16 weeks and at 4-week intervals thereafter.

OPHTHALMOSCOPIC EXAMINATION: Yes / No / No data
- Time schedule for examinations: Pre-test and during Week 52.
- Dose groups that were examined: All dogs

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Pre-test and in Weeks 14, 27 and 53
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes, overnight prior to and during sampling
- How many animals: All dogs

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Pre-test and in Weeks 14, 27 and 53
- Animals fasted: Yes, overnight prior to and during sampling
- How many animals: All dogs

URINALYSIS: Yes
- Time schedule for collection of urine: Pre-test and in Weeks 14, 27 and 53
- Animals fasted: Yes, overnight prior to and during sampling
- How many animals: All dogs

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

Variance homogeneity was analysed by Levene’s test. One-way ANOVA was performed and, if significant, Dunnett’s t-test was performed to compare treated groups with the control group.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

See Table 1

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

See "Details on results"

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

All animals survived to the end of the treatment period and no treatment-related clinical signs of toxicity or ocular defects occurred at any dose level. There was a treatment-related decrease in the overall body weight gains of both sexes at 16000ppm and of females at 3200ppm (Table 1). Body weight gains were 30.0 - 37.1% lower than the controls and statistically significant (p < 0.05) in the female groups. The body weight gains of males at 3200ppm and both sexes at 640ppm were unaffected by treatment. Females treated at 3200 and 16000ppm showed 8.0 and 12.3% decreases, respectively, in food consumption, but consumption was unaffected by treatment in the other groups. Water consumption was unaffected by treatment at all dose levels.

There were no treatment-related effects at any dose level on the hematological, serum clinical chemistry and urinalysis profiles. The only statistically significant differences between the 16000ppm group and the controls were higher urine pH at week 27 and slightly higher serum albumin and potassium ion concentrations at week 53 in females. These differences are considered incidental to treatment with dinotefuran because they were not evident at other sampling intervals and were not associated with correlative clinical or histopathological alterations. There were no treatment-related effects at any dose level on the incidence of macroscopic findings at necropsy or on organ weights and ratios. An apparent, treatment-related effect on the group mean thymus weight of all male treated groups was considered not to be an effect of treatment on comparison with laboratory historical control data (HCD) which showed only one low dose and one high dose animal with values outside the HCD range (see historical data, attached).

Statistically significant (p < 0.05) higher uterus/body weight ratios in females treated at 3200ppm and higher ovary/body weight ratios in females at 3200 and 16000ppm were attributed to lower body weights at termination. There were no treatment-related histopathological alterations at any dose level. All microscopic findings were typical of those occurring spontaneously in beagle dogs.

Dose descriptor:

NOEL

Effect level:

3 200 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Equivalent to a dose level of 111mg/kg bw/day. Based on the occurrence of growth retardation in males at 16000ppm.

Dose descriptor:

NOEL

Effect level:

640 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Equivalent to a dose level of 22mg/kg bw/day. Based on the occurrence of growth retardation and reduced food consumption in females at 3200 and 16000ppm.

Dose descriptor:

NOAEL

Effect level:

16 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Equivalent to dose levels of 559mg/kg bw/day (males) and 512mg/kg bw/day (females). Based on the absence of correlative clinical and pathological alterations associated with the observed growth retardation.

Critical effects observed:

not specified

Table 1: Treatment related effects on bodyweight gain and food consumption

Treatment	Group mean body weight (kg) in week:					Overall weight	Mean food
(ppm)	1	14	28	40	52	gain (kg)	intake (g/day)
Males:
0	8.7	11.4	11.6	11.7	11.7	3.0	349
640	8.7	11.4	11.7	11.9	11.6	2.9	330
3200	8.9	11.0	11.5	12.1	11.9	3.0	368
16000	8.5	11.0	10.7	11.1	10.6	2.1	363
Females:
0	8.0	10.8	11.3	12.0	11.5	3.5	350
640	7.9	10.2	10.6	11.1	11.4	3.5	348
3200	7.9	9.9*	10.3	10.4*	10.1*	2.2*	322
16000	7.9	9.7*	10.2	10.2*	10.2*	2.3*	307

* p < 0.05

Conclusions:

No target organs were identified in either sex at the highest dose level employed. The no-observed-effect-level (NOEL) for all effects was established as 3200ppm diet (males) and 640ppm (females), equivalent to a dose level of 111mg/kg bw/day (males) and 22mg/kg bw/day (females), based on the occurrence of growth retardation in males at 16000ppm and growth retardation and reduced food consumption in females at 3200 and 16000ppm.
The no-observed-adverse-effect-level (NOAEL) is considered to be 16000ppm in both sexes, equivalent to dose levels of 559mg/kg bw/day (males) and 512mg/kg bw/day (females), based on the absence of correlative clinical and pathological alterations associated with the observed growth retardation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

22 mg/kg bw/day

Study duration:

subchronic

Species:

dog

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: inhalation, other

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09/10/2001 - 26/02/2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP, Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

(1981)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:WI(GlxBRL/Han)BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: About 9 weeks old
- Weight at study initiation: 173.8-226.6 g for males. 144.9-182.2 g for females

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: The calculated MMAD ± GSD were 2.03 ± 3.31 µm, 1.80 ± 3.60 µm and 1.55 ± 2.96 µm, respectively.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE
- Exposure apparatus: Wright dust feed generator connected to a 40L-exposure chamber utilising a tangential, continuous air-flow system.
- See Table 7.5.2-1

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- The actual exposure concentrations were measured gravimetrically at least hourly during each exposure period for each concentration.
- The particle size of the atmospheres was determined gravimetrically pre-exposure and then once a week throughout the exposure period. The particle size of the highest atmosphere concentration was measured on a further 3 occasions during Week 1.
- Test atmospheres were sampled for up to 30 seconds by drawing through a 9-stage cascade impactor (0.52 - 100µm)
- The mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) were calculated for each occasion.

Duration of treatment / exposure:

6 hours per day

Frequency of treatment:

Daily for 29 or 30 days

Remarks:

Doses / Concentrations:
2.89 mg/L
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
16.02 mg/L
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
61.24 mg/L
Basis:
nominal conc.

No. of animals per sex per dose:

10 rats per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The target atmosphere chamber concentrations were selected by the Sponsor based on findings on a preceding 6 day prelliminary study. The highest exposure concentration (2 mg/L) was selected as this was considered to be the highest practical concentration obtainable in the respirable range and would result in a classification of Category IV in the single 4-hour exposure study (EPA OPPTS 870.1000 guideline). The intermediate and low exposure concentrations are fractions of the high exposure atmosphere concentration.
- Post exposure observation period: none

Positive control:

None

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The animals were observed twice daily for morbidity and mortality. Animal observation following exposure was performed daily, generally immediately after exposure and several times up to 2 hours after exposure.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded before exposure on Day 1, at weekly intervals and at necropsy.

FOOD CONSUMPTION:
- Time schedule for examinations: Food consumption was determined weekly.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmoscopic examinations were performed on all animals pre-exposure and on all animals treated at 0 or 2.08mg/L during Week 4.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: During Week 4
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes, overnight prior to sampling
- How many animals: All rats

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: During Week 4
- Animals fasted: Yes, overnight prior to sampling
- How many animals: All rats

URINALYSIS: Yes
- Time schedule for collection of urine: During Week 4
- Animals fasted: Yes, overnight prior to sampling
- How many animals: All rats

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

Where appropriate, data were subjected to statistical analysis using one-way ANOVA. Levene’s test was used to evaluate the homogeneity of variance. Where data were not heterogeneous Dunnett’s test was employed. Clinical chemistry data were analysed using non-parametric tests, Kruskal-Wallis ANOVA and the Terpstra-Jonckheere test. Organ weight data was analysed using ANCOVA followed by Dunnett’s test.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

There were no deaths or treatment-related clinical signs in any exposure group. The body weight gains of all male treated groups were reduced during week 1. Since the overall effect on male body weight gain was minimal and transient, and associated with a slight decrease in food consumption, it is considered not to be an adverse effect. The weight gain of all female treated groups was comparable to the control values throughout the study. The mean weekly food consumption of all male treated groups in weeks 1 and 2, and in the high dose group in week 3, was slightly lower than the control consumption. However, the differences were not statistically significant. The food consumption of all female treated groups was unaffected by exposure to dinotefuran.
There were no treatment-related ophthalmological findings at the highest exposure concentration. There were no treatment-related effects on the hematological and plasma and urine clinical chemistry profiles in either sex at any exposure concentration. There were no treatment-related macroscopic findings, organ weight changes or histopathological alterations.
No target organs were identified in either sex at the highest dose level employed.

See Table 1.

Key result

Dose descriptor:

NOAEL

Effect level:

2.08 mg/L air

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Critical effects observed:

not specified

Table A6.3.3-1   Summary of body weight gain and food consumption

Study interval	Sex	Group mean body weight gain (g) at (mg/L):
		0	0.22	0.66	2.08
Week 1	Male	26.7	17.1*	16.0**	14.3**
Week 2 - week 4		38.2	35.4	35.2	34.0
Overall (week 1 - week 4)		64.8	52.5	51.1*	48.3**
Week 1	Female	8.2	7.2	7.1	7.0
Week 2 - week 4		17.8	19.8	14.1	22.5
Overall (week 1 - week 4)		26.0	27.0	21.2	29.5
		Group mean food consumption (g/week):
Week 1	Male	146.3	137.8	138.9	130.7
Week 2		155.1	148.4	147.0	140.0
Week 3		155.3	153.9	151.3	145.7
Week 4		134.7	137.3	138.9	135.9
Week 1	Female	109.5	110.1	109.0	109.7
Week 2		113.5	117.6	114.2	117.9
Week 3		117.2	122.8	119.2	120.2
Week 4		110.7	113.1	114.4	119.8

 

Conclusions:

A no-observed-adverse-effect-level for respirable dinotefuran was established in both sexes as 2.08 mg/L, the maximum technically achievable aerosol concentration with a MMAD ± GSD of 1.55 ± 2.96 µm.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

2 008 mg/m³

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: inhalation, other

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09/10/2001 - 26/02/2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP, Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

(1981)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:WI(GlxBRL/Han)BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: About 9 weeks old
- Weight at study initiation: 173.8-226.6 g for males. 144.9-182.2 g for females

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: The calculated MMAD ± GSD were 2.03 ± 3.31 µm, 1.80 ± 3.60 µm and 1.55 ± 2.96 µm, respectively.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE
- Exposure apparatus: Wright dust feed generator connected to a 40L-exposure chamber utilising a tangential, continuous air-flow system.
- See Table 7.5.2-1

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- The actual exposure concentrations were measured gravimetrically at least hourly during each exposure period for each concentration.
- The particle size of the atmospheres was determined gravimetrically pre-exposure and then once a week throughout the exposure period. The particle size of the highest atmosphere concentration was measured on a further 3 occasions during Week 1.
- Test atmospheres were sampled for up to 30 seconds by drawing through a 9-stage cascade impactor (0.52 - 100µm)
- The mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) were calculated for each occasion.

Duration of treatment / exposure:

6 hours per day

Frequency of treatment:

Daily for 29 or 30 days

Remarks:

Doses / Concentrations:
2.89 mg/L
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
16.02 mg/L
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
61.24 mg/L
Basis:
nominal conc.

No. of animals per sex per dose:

10 rats per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The target atmosphere chamber concentrations were selected by the Sponsor based on findings on a preceding 6 day prelliminary study. The highest exposure concentration (2 mg/L) was selected as this was considered to be the highest practical concentration obtainable in the respirable range and would result in a classification of Category IV in the single 4-hour exposure study (EPA OPPTS 870.1000 guideline). The intermediate and low exposure concentrations are fractions of the high exposure atmosphere concentration.
- Post exposure observation period: none

Positive control:

None

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The animals were observed twice daily for morbidity and mortality. Animal observation following exposure was performed daily, generally immediately after exposure and several times up to 2 hours after exposure.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded before exposure on Day 1, at weekly intervals and at necropsy.

FOOD CONSUMPTION:
- Time schedule for examinations: Food consumption was determined weekly.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmoscopic examinations were performed on all animals pre-exposure and on all animals treated at 0 or 2.08mg/L during Week 4.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: During Week 4
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes, overnight prior to sampling
- How many animals: All rats

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: During Week 4
- Animals fasted: Yes, overnight prior to sampling
- How many animals: All rats

URINALYSIS: Yes
- Time schedule for collection of urine: During Week 4
- Animals fasted: Yes, overnight prior to sampling
- How many animals: All rats

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

Where appropriate, data were subjected to statistical analysis using one-way ANOVA. Levene’s test was used to evaluate the homogeneity of variance. Where data were not heterogeneous Dunnett’s test was employed. Clinical chemistry data were analysed using non-parametric tests, Kruskal-Wallis ANOVA and the Terpstra-Jonckheere test. Organ weight data was analysed using ANCOVA followed by Dunnett’s test.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

There were no deaths or treatment-related clinical signs in any exposure group. The body weight gains of all male treated groups were reduced during week 1. Since the overall effect on male body weight gain was minimal and transient, and associated with a slight decrease in food consumption, it is considered not to be an adverse effect. The weight gain of all female treated groups was comparable to the control values throughout the study. The mean weekly food consumption of all male treated groups in weeks 1 and 2, and in the high dose group in week 3, was slightly lower than the control consumption. However, the differences were not statistically significant. The food consumption of all female treated groups was unaffected by exposure to dinotefuran.
There were no treatment-related ophthalmological findings at the highest exposure concentration. There were no treatment-related effects on the hematological and plasma and urine clinical chemistry profiles in either sex at any exposure concentration. There were no treatment-related macroscopic findings, organ weight changes or histopathological alterations.
No target organs were identified in either sex at the highest dose level employed.

See Table 1.

Key result

Dose descriptor:

NOAEL

Effect level:

2.08 mg/L air

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Critical effects observed:

not specified

Table A6.3.3-1   Summary of body weight gain and food consumption

Study interval	Sex	Group mean body weight gain (g) at (mg/L):
		0	0.22	0.66	2.08
Week 1	Male	26.7	17.1*	16.0**	14.3**
Week 2 - week 4		38.2	35.4	35.2	34.0
Overall (week 1 - week 4)		64.8	52.5	51.1*	48.3**
Week 1	Female	8.2	7.2	7.1	7.0
Week 2 - week 4		17.8	19.8	14.1	22.5
Overall (week 1 - week 4)		26.0	27.0	21.2	29.5
		Group mean food consumption (g/week):
Week 1	Male	146.3	137.8	138.9	130.7
Week 2		155.1	148.4	147.0	140.0
Week 3		155.3	153.9	151.3	145.7
Week 4		134.7	137.3	138.9	135.9
Week 1	Female	109.5	110.1	109.0	109.7
Week 2		113.5	117.6	114.2	117.9
Week 3		117.2	122.8	119.2	120.2
Week 4		110.7	113.1	114.4	119.8

 

Conclusions:

A no-observed-adverse-effect-level for respirable dinotefuran was established in both sexes as 2.08 mg/L, the maximum technically achievable aerosol concentration with a MMAD ± GSD of 1.55 ± 2.96 µm.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

2 008 mg/m³

Study duration:

subacute

Species:

rat

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Remarks:

other: repeated dose

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22/01/2001 - 12/10/2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP, Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Version / remarks:

1981

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.9 (Repeated Dose (28 Days) Toxicity (Dermal))

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3200 (Repeated Dose Dermal Toxicity -21/28 Days)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:CD®(SD)IGS BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

Age at study initiation: about 8 weeks old
Weight at study initiation: 247-326 g for males. 166-218 g for females

Type of coverage:

semiocclusive

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5%

Details on exposure:

Area covered: 10 % of body surface area
Total volume applied: 2 mL/ kg dinotefuran

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis for the concentration of test material in the dose preparations were conducted using analytical method MP-MT47-MA, supplied by the Sponsor and validated by the laboratory.

Duration of treatment / exposure:

Duration of treatment: 28 days

Frequency of treatment:

Frequency of exposure: 7 days per week
Duration of exposure 6-7 hours per day

Remarks:

Doses / Concentrations:
0 (vehicle only), 40, 200 and 1000mg/kg bw/day
Basis:
nominal per unit body weight

No. of animals per sex per dose:

10 males and 10 females per group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The goal of dose selection was to achieve a gradient of toxic effects. Dose levels selected were based on preliminary results of a previous 14-day range finding dermal toxicity study with dinotefuran in rats. Dermal application on dinotefurn had no apparent effects on macroscopic or microscopic pathology findings. Based on the lack of findings, the high-dose level was considered to be the limit dose.
- Post exposure observation period: none

Positive control:

None

Observations and examinations performed and frequency:

Clinical signs: Yes, on days 1, 8, 15, 22 and 29

Mortality: Yes, on days 1, 8, 15, 22 and 29

Body weight: Yes, pre-dose and weekly thereafter starting on the first day of treatment

Food consumption : Yes, weekly

Water consumption : No

Ophthalmoscopic examination: Yes, pre-dose and on day 26

Haematology: Yes
Number of animals: all animals
Time points: end of study
Parameters: Haematocrit, haemoglobin concentration, erythrocyte count, total and differential leukocyte count, platelet count, clotting time, prothrombin time, thromboplastin time

Clinical Chemistry: Yes
Number of animals: all animals
Time points: end of study
Parameters: sodium, potassium, glucose, total cholesterol, urea, blood urea nitrogen, total bilirubin, creatinine, total protein, albumin, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma glutamyl transpeptidase, sorbitol dehydrogenase, methaemoglobin, lipids, hormone (specify hormones), acid/base balance, cholinesterase inhibition.

Urinalysis:No

Sacrifice and pathology:

Organ Weights: Yes
Organs: liver, kidneys, adrenals, testes, epididymides, uterus, ovaries, thymus, spleen, brain, heart

Gross and histopathology: Yes
High dose group and controls
Organs: brain, spinal cord, pituitary, thyroid, parathyroid, thymus, oesophagus, salivary glands, stomach, small and large intestines, liver, pancreas, kidneys, adrenals, spleen, heart, trachea, lungs, aorta, gonads, uterus, female mammary gland, prostate, urinary bladder, gall bladder (mouse), lymph nodes peripheral nerve, bone marrow, skin, eyes.

The decedent was also subjected to necropsy, but organ weights were not recorded.

Other examinations:

Dermal irritation reactions were scored immediately before application on days 1, 8, 15, 22, 29 and on the day of necropsy. Erythema, edema, atonia, desquamation and fissuring reactions were scored on a 4-point scale from 0 (none) to 3 (severe). The occurrence of eschar and exfoliation were also recorded.

Statistics:

Where appropriate, Levene’s test was used to test homogeneity of variance. One-way ANOVA was applied to appropriate data followed by Dunnett’s t-test if ANOVA was significant.

Clinical signs:

no effects observed

Dermal irritation:

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

Treatment-related histopathological alterations at 1000mg/kg bw/day were confined to a minimal increase in the incidence and severity of acanthosis / hyperkeratosis in the treated skin of females. The finding is considered not to be an adverse effect or toxicologically relevant because slight to moderate acanthosis / hyperkeratosis occurred in 2 control females and in the untreated skin of a female at 200mg/kg bw/day. Furthermore, all control and 1000mg/kg bw/day males also showed the skin alteration. There were no treatment-related histopathological alterations in the other tissues examined from animals treated at 1000mg/kg bw/day.

See Table 1.

There were no treatment-related effects at any dose level on any of the ECO parameters evaluated and no statistically significant (p > 0.05) effects on quantitative motor activity. One male treated at 40mg/kg bw/day and 2 females at 1000mg/kg bw/day showed slight (grade 1) skin atonia at the application site on one or two occasions during the treatment period. There were no other signs of dermal irritation at any dose level. Since the observed atonia was transient and was not accompanied by other signs of irritation, the finding is considered not to be an adverse effect.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on the absence of systemic and local adverse effects at this dose level.

Critical effects observed:

not specified

Table 1:   Incidence of selected histopathological alterations

Organ:	Incidence of lesion in:
finding	Males treated at (mg/kg bw/day):				Females treated at (mg/kg bw/day):
	0	40	200	1000	0	40	200	1000
Treated skin: - no. examined - inflammation -acanthosis/   hyperkeratosis	10 2   10	1 0   1	0 0   0	10 2   10	10 3   2	0 0   0	0 0   0	10 2   8
Untreated skin: - no. examined - inflammation - fibrosis - ulceration - acanthosis/ hyperkeratosis	10 8 0 0   0	1 0 0 0   0	0 0 0 0   0	10 4 0 0   0	10 8 0 0   1	0 0 0 0   0	1 1 1 0   1	10 7 0 1   0

 

Conclusions:

A no-observed-adverse-effect-level (NOAEL) was established as > 1000mg/kg bw/day, based on the absence of systemic and local adverse effects at this dose level.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat