spirodiclofen (ISO);3-(2,4-dichlorophenyl)-2-oxo-1-oxaspiro[4.5]dec-3-en-4-yl 2,2-dimethylbutyrate

Repeated dose toxicity: oral

Administrative data

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2001

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 452 (Chronic Toxicity Studies)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.4100 (Chronic Toxicity)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

3-(2,4-Dichlorophenyl)-2-oxo-1-oxaspiro[4.5]dec-3-en-4-yl 2,2-dimethylbutanoate

Cas Number:

148477-71-8

Molecular formula:

C21H24Cl2O4

IUPAC Name:

3-(2,4-Dichlorophenyl)-2-oxo-1-oxaspiro[4.5]dec-3-en-4-yl 2,2-dimethylbutanoate

Test animals

Species:

dog

Strain:

Beagle

Details on species / strain selection:

Standard laboratory species/strain

Sex:

male/female

Details on test animals or test system and environmental conditions:

40 purebred beagles (20 males, 20 nulliparous non-pregnant females) from the breeder Harlan-Winkelmann were used in the study. After an acclimatization period, dogs judged to be healthy both clinically and on the basis of the laboratory data from the screening investigation (from a total group of 25 males and 25 females) were randomly assigned to groups after prior stratification for sex and body weight. In week -1 the dogs were 33 - 37 weeks old and weighed between 10.4 and 15.7 kg. During acclimatization and the conduct of the study all the male
and female beagles were housed in individual cages (floor area approximately 1.10 x 1.15 m) in a room reserved for the sole use of the present study. The room was force-ventilated. The temperature, which was recorded, was predominantly 19.0 - 26.0° C during the study. The average atmospheric humidity was approximately 17 - 95 %. Brief fluctuations in the environmental conditions (temperature, humidity), that could occur, for example, when the room and the cages were being spray-cleaned, had no clinically discernible influence on the health of the animals. The premises were illuminated by diffuse daylight (skylights), but mainly by fluorescent lamps, which regulated the day/night cycle automatically (12 h day and 12 h night). Except on weekends and on public holidays all animals, separated according to sex, were allowed to exercise daily for at least 60 min - generally in the morning - in a special room directly
connected to the animal room.

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

Test material was mixed with feed at different concentrations and consumed daily.

Vehicle:

unchanged (no vehicle)

Remarks:

The test material was administered in the diet

Details on oral exposure:

Groups of 4 male and 4 female Beagle dogs were treated orally over a period of about 52 weeks with daily concentrations of 0 (control group), 20, 50, 150 and 600 ppm spirodiclofen in their diet. The highest dose group was initially treated with 500 ppm until study week 4, after which this dose group received 600 ppm in agreement with EPA.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before starting the study, it had been found out that the feed/substance mix was not stable over a period of 7 days. The analytical data verify that the test material in the animal ration was homogeneously distributed within the concentration range of 400 ppm to 10000 ppm. Under current sample preparation and handling conditions comparable to those in the actual study the chemical stability was assured for a period of at least 4 days. The stability in moist animal feed within the concentration range of 100 ppm to 10000 ppm was assured for a period of at least 5 hours. In addition, stability in moist feed was assured for the concentrations of 20 and 50 ppm for at least 2 hours.

Duration of treatment / exposure:

52 weeks

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Remarks:

Controls

Doses / concentrations 2

Dose / conc.:

20 ppm

Remarks:

Equivalent to a mean intake of 0.57 mg/kg bw/d (sexes combined)

Doses / concentrations 3

Dose / conc.:

50 ppm

Remarks:

Equivalent to a mean intake of 1.45 mg/kg bw/d (sexes combined)

Doses / concentrations 4

Dose / conc.:

150 ppm

Remarks:

Equivalent to a mean intake of 4.54 mg/kg bw/d (sexes combined)

Doses / concentrations 5

Dose / conc.:

600 ppm

Remarks:

Equivalent to a mean intake of 16.9 mg/kg bw/d (sexes combined)

No. of animals per sex per dose:

Groups of 4 male and 4 females per dose

Control animals:

yes, plain diet

Details on study design:

Groups of 4 male and 4 female Beagle dogs were treated orally over a period of about 52 weeks with daily concentrations of 0 (control group), 20, 50, 150 and 600 ppm spirodiclofen in their diet. The highest dose group was initially treated with 500 ppm until study week 4, after which this dose group received 600 ppm in agreement with EPA. The test substance intakes over the whole study period were 0.57, 1.45, 4.54, 16.9 mg/kg bw/day (mean values for both sexes). The animals were observed daily for clinical signs. Testing of reflexes (pupillary, corneal, patellar, extensor, postural and flexor), body temperature, pulse rate and heart rate, and ECG determinations were performed in weeks -3, 6, 12, 26, 39 and 52 with the following exceptions: heart rates were performed in week 27 rather than week 26 and reflexes were not tested during the week 26/27 interval. Clinical chemistry and hematology measurements were done in weeks –3, -1, 3, 6, 12, 26, 39 and 52.

Positive control:

Not required for this study type.

Examinations

Observations and examinations performed and frequency:

The animals were observed daily for clinical signs. Testing of reflexes (pupillary, corneal, patellar, extensor, postural and flexor), body temperature, pulse rate and heart rate, and ECG determinations were performed in weeks -3, 6, 12, 26, 39 and 52 with the following exceptions: heart rates were performed in week 27 rather than week 26 and reflexes were not tested during the week 26/27 interval.

Sacrifice and pathology:

Clinical chemistry and hematology measurements were done in weeks –3, -1, 3, 6, 12, 26, 39 and 52.

Results and discussion

Results of examinations

Clinical signs:

no effects 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:

effects observed, treatment-related

Description (incidence and severity):

The clinical chemical investigations revealed a slight increase of transaminase activities (ALT, AST) in the 600 ppm males and females but also in control animals beginning in study week 6 so that a treatment-relationship is not obvious. Cholesterol levels were slightly decreased at 600 ppm in both sexes in week 6 and 12.

Endocrine findings:

not examined

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Increased relative organ weights of testes and prostates in 150 and 600 ppm males and of the adrenal glands in 600 ppm females were observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathology revealed minimal or slight Leydig cell vacuolation in the testes of all animals dosed at 600 ppm and in one male of this group also slight Leydig cell hypertrophy. In one male from this group a bilateral slight tubular degeneration was observed. 

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Evidence of microsomal liver enzyme induction was seen at 600 ppm. Slight elevations of NDE were also seen at lower doses but ODE was only increased at 600 ppm. Since no hepatocellular hypertrophy as a morphological correlate of liver enzyme induction occurred at any dose this response is regarded as equivocal or borderline effect.

Details on results:

General observations: No clinical signs were observed, and all animals survived the study period. Determinations of reflexes, body temperature, pulse rate and heart rate did not reveal substance-induced changes. Also the ECG measurements and the ophthalmoscopic examinations did not indicate treatment-related effects. No significant differences could be observed regarding the nutritional state of the animals. Feed intake was not impaired by the treatment in any dose groups, an incomplete feed intake in the initial study phase occurred in all groups without a dose-relationship. Based on the dietary consumption, the test compound intake was calculated. Body weight development was normal in all treatment groups as compared to the control group.
Hematology, clinical chemistry, urinalysis: Hematological investigations did not reveal any remarkable differences between control and test animals. The clinical chemical investigations revealed a slight increase of transaminase activities (ALT, AST) in the 600 ppm males and females but also in control animals beginning in study week 6 so that a treatment-relationship is not obvious. Cholesterol levels were slightly decreased at 600 ppm in both sexes in week 6 and 12. Urinalysis did not reveal any changes between control and treated animals.
Evidence of microsomal liver enzyme induction was seen at 600 ppm. Slight elevations of NDE were also seen at lower doses but ODE was only increased at 600 ppm. Since no hepatocellular hypertrophy as a morphological correlate of liver enzyme induction occurred at any dose this response is regarded as equivocal or borderline effect Gross pathology, organ weights, histopathology: At necropsy no evidence of any gross pathological findings related to treatment with the test compound was found. Increased relative organ weights of testes and prostates in 150 and 600 ppm males and of the adrenal glands in 600 ppm females were observed. Histopathology revealed minimal or slight Leydig cell vacuolation in the testes of all animals dosed at 600 ppm and in one male of this group also slight Leydig cell hypertrophy. In one male from this group a bilateral slight tubular degeneration was observed.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

50 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: adrenal effects (vacuolization

Target system / organ toxicity

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

50 ppm

Organ:

adrenal glands

Any other information on results incl. tables

Chronic toxicity dog study: Clinical Chemistry (males)

Week	AST (U/L)	ALT(U/L)	Chol(mmol/L)
0ppm
-3	11.0	17.5	3.93
-1	11.0	19.1
3	11.7	20.1	3.29
6	13.4	19.1	3.16
12	14.9	19.8	2.75
26	13.4	24.5	3.26
39	12.0	37.6	3.55
52	18.6	29.7	3.28
20 ppm
-3	10.4	16.4	4.62
-1	11.2	15.4
3	12.6	20.2	3.75
6	18.0	26.3	3.13
12	13.5	18.8	3.30
26	13.6	23.2	4.26
39	12.7	21.2	4.54
52	15.0	26.3	4.82
50ppm
-3	11.6	17.2	4.84
-1	10.5	15.6
3	14.5	20.1	3.79
6	14.2	17.4	3.62
12	12.6	19.1	3.90
26	13.9	20.1	4.07
39	13.8	21.3	4.20
52	14.3	21.4	4.09

Chronic toxicity dog study: Clinical Chemistry (females)

week	AST	ALT	Chol
0mm
-3	12.3	14.8	3.93
-1	10.2	13.0
3	11.9	13.6	3.88
6	12.8	20.6	3.16
12	15.3	20.1	2.73
26	14.8	24.6	4.39
39	11.1	83.5	5.90
52	11.0	16.0	5.89
20ppm
-3	12.3	15.8	3.76
-1	10.6	14.3
3	11.1	15.4	3.18
6	19.4	18.0	2.77
12	12.4	17.0	3.33
26	15.3	20.5	4.81
39	10.6	16.5	4.31
52	12.2	16.0	4.58
50ppm
-3	13.9	19.5	4.09
-1	12.7	16.8
3	17.2	22.5	3.26
6	25.1	25.2	2.73
12	14.4	20.3	3.08
26	15.8	18.9	3.88
39	11.7	18.4	4.75
52	13.4	18.5	4.09

Chronic toxicity dog study: Clinical Chemistry (females) (continued)

week	AST	ALT	Chol
150ppm
-3	13.7	18.6	4.12
-1	12.1	17.2
3	14.7	18.5	3.05
6	16.9	21.0	2.82
12	14.1	18.8	2.87
26	13.3	22.1	3.47
39	12.0	20.0	3.57
52	12.7	17.2	5.54
600ppm
-3	12.4	16.3	3.99
-1	11.4	15.5
3	15.7	16.7	3.07
6	20.5	28.1	2.67
12	15.6	21.7	3.04
26	15.9	20.5	4.21
39	13.2	18.9	4.44
52	14.0	18.9	4.77

Chronic study in dogs: microsomal liver enzyme activities (both sexes)

Liver enzymes	Dose (ppm)
	0	20	50	150	600
N-DEM (1)	71.1	73.7	85.5	100.1*	144.00**
0-DEM (1)	20.4	20.1	23.2	23.7	32.2
P450 (2)	27.6	27.3	29.8	30.1	34.4*

1 mU/g
2 nmol/g

Chronic toxicity dog study: Relative organ weights (g/kg bw)

Dose (ppm)	0	20	50	150	600
Males
Testes	1.38	1.64	1.44	1.77	1.79
Prostate	0.70	0.71	0.72	0.73	0.83
Females
Adrenals	0.131	0.140	0.130	0.145	0.154

Applicant's summary and conclusion

Conclusions:

The NOAEL of 50 ppm (equivalent to 1.38 mg/kg bw/d in males and of 1.52 mg/kg bw/d in females) was based on adrenal effects (vacuolization) at 150 ppm.

Executive summary:

Groups of 4 male and 4 female Beagle dogs were treated with spirodiclofen orally over a period of  52 weeks using dietary concentrations of 0 (control group), 20, 50, 150 and 600 ppm. The highest dose group was initially treated with 500 ppm until study week 4, after which this dose group received 600 ppm. The test substance intakes over the whole study period were 0.57, 1.45, 4.54, 16.9 mg/kg bw/d (mean values for both sexes). The animals were daily observed for clinical  signs. Testing of reflexes (pupillary, corneal, patellar, extensor, postural and flexor), body temperature, pulse rate and heart rate and ECG determinations were performed in weeks -3, 6, 12, 26, 39 and 52. Clinical chemistry and hematology measurements were done in weeks –3, -1, 3, 6, 12, 26, 39 and 52.  No clinical signs were observed, and all animals survived the study period.  Determinations of reflexes, body temperature, pulse rate and heart rate did not reveal substance-induced changes. ECG measurements and the ophthalmoscopic examinations did not indicate treatment-related effects. No significant differences could be observed regarding the nutritional state of the animals. Feed intake was not impaired by the treatment in any dose groups, an incomplete feed intake in the initial study phase occurred in all groups without a dose-relationship. Body weight development was normal in all treatment groups as compared to the control group. Hematological investigations did not eveal any remarkable differences between control and test animals. The clinical chemical investigations revealed a slight increase of transaminase activities (ALT, AST) in the 600 ppm males and females but also in control animals beginning in study week 6 so that a treatment-relationship is not obvious. Cholesterol levels were slightly decreased at 600 ppm in both sexes in week 6 and 12. Urinalysis did not reveal any changes between control and treated animals. Evidence of microsomal liver enzyme induction was seen at 600 ppm. Slight elevations of NDE were also seen at lower doses but ODE was only increased at 600 ppm. Since no hepatocellular hypertrophy as a morphological correlate of liver enzyme induction occurred at any dose this response is regarded as equivocal or borderline effect.  At necropsy, no evidence of any gross pathological findings related to treatment was found. Increased relative organ weights of testes and prostates in 150 and 600 ppm males and of the adrenal glands in 600 ppm females were observed. Histopathology revealed minimal or slight Leydig cell vacuolation in the testes of all animals dosed at 600 ppm and in one male of this group also slight Leydig cell hypertrophy. In one male from this group a bilateral slight tubular degeneration was observed.  The NOAEL of 50 ppm (equivalent to 1.38 mg/kg bw/d in males and of 1.52 mg/kg bw/d in females) was based on adrenal effects (vacuolization) at 150 ppm.