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

Endpoint summary

Description of key information

The Key Studies for the repeated dose toxicity endpoint are as follows:

90-day oral (dietary) rat study (M-005766-02-1)

12-month oral (dietary) dog study (M-045424-02-1)

28-day dermal toxicity study in the rat (M-011043-01-1)

A number of Supporting Studies are also available:

28-day oral (dietary) rat study (M-024721-01-1)

28-day oral (dietary) dog study (M-027510-02-1)

8-week investigative oral (dietary) dog study (M-052495-01-1)

90-day oral (dietary) mouse study (M-005729-03-1)

90-day oral (dietary) dog study (M-032514-02-1)

Non-standard 19-week oral (dietary) rat study (M-049165-01-1)

Chronic toxicity/carcinogenicity studies are also available in the rat (M-026284-02-1) and in the mouse (M-044116-01-1).

The lowest NOAEL (1.45 mg/kg bw/d) is reported in the 12-month oral (dietary) dog study (M-045424-02-1); however, the mouse chronic toxicity/carcinogenicity study does not identify a NOAEL (LOAEL =4.1 mg/kg bw/d).

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 452 (Chronic Toxicity Studies)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.4100 (Chronic Toxicity)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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.

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

Dose / conc.:

0 ppm

Remarks:

Controls

Dose / conc.:

20 ppm

Remarks:

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

Dose / conc.:

50 ppm

Remarks:

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

Dose / conc.:

150 ppm

Remarks:

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

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.

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.

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.

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

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

50 ppm

Organ:

adrenal glands

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

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

1.45 mg/kg bw/day

Study duration:

chronic

Species:

dog

Quality of whole database:

A comprehensive and high-quality database of repeated dose oral toxicity studies of up to chronic duration are available for the rat, mouse and dog. A sub-acute dermal toxicity study is also available for the rat. The key studies are GLP- and guideline-compliant; the supporting studies include guideline-compliant and non-guideline investigative studies.

System:

endocrine system

Organ:

adrenal glands

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

9 February 1998 to 4 May 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: 67/548/EEC

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Standard laboratory species/strain with background control data available

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 7-8 weeks
- Weight at study initiation: 223-250 g (M), 200-207 g (F)
- Fasting period before study: none
- Housing: individual
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least five days

DETAILS OF FOOD AND WATER QUALITY:
The rations consisted of fixed-formula standard diet (Altromin 1324 (= pelletized 1321) Diet for Rats and Mice) supplied by Altromin GmbH, and of tap water (drinking bottles). Feed and water were available ad libitum and were supplied in polycarbonate bottles and in stainless steel cage cover, respectively. The nutritive composition and the contaminant-content of the standard diet were routinely spot-checked and analyzed. The tap water complied with the German Drinking Water Ordinance.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-22
- Humidity (%): 50-60
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 18 February 1998 To: 17 March 1998

Type of coverage:

semiocclusive

Vehicle:

unchanged (no vehicle)

Remarks:

The test material was applied as a solid onto a wet gauze pad

Details on exposure:

Two days before the start of the treatment the back and flanks of the rats were shorn. Any regrowth of hair was shaved off the skin areas marked for treatment twice weekly. For each animal the required amount of test substance was weighed and transferred to the wet gauze-layer (5.5 x 5.5 cm = 30.25 cm2) of a sterile patch and placed on the rat's back. The gauze strip was secured in place using adhesive stretch tape (8 x 23 cm). Additionally, the mobility of the rats was impaired by a rat jacket. The gauze strip has the same size as the application area, i.e. 30.25 cm2. Thus, the application area was greater than 10% of the body surface area of the rat. After an exposure time of 6 hours the fixing bandage and the gauze strip were removed and the treated area cleaned with soap and water. Control animals were treated with tap water.

Analytical verification of doses or concentrations:

no

Remarks:

Analytical determinations of stability and homogeneity were not performed because the test substance was applied undiluted.

Duration of treatment / exposure:

The test material was applied for 6 hours/day

Frequency of treatment:

The test material was applied daily; 5 days/week for 28 days (total 22 applications). On the first three weeks of the study, the formulations were applied on 5 days per week, and in the fourth week also on Saturday and Sunday. Thus, both males and females were dosed daily over the final seven days prior to terminal sacrifice.

Dose / conc.:

0 mg/kg bw/day

Remarks:

Control; water only

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Test material moistened with water

No. of animals per sex per dose:

Groups of 5 male and 5 female rats per dose.

Control animals:

yes, concurrent no treatment

Details on study design:

Groups of 5 male and 5 female Wistar rats of the strain Hsd Cpb: WU were administered spirodiclofen, at levels of 0 and 1000 mg/kg bw/d by dermal application. Analytical determinations of stability and homogeneity were not performed because the test substance was applied undiluted and only moistened with water immediately before application. Males and females received 22 applications within a total period of 28 days. The exposure time per day was 6 hours. The doses were based on the results of a dose range finding study in female rats. In that study, the doses were 0 and 1000 mg/kg bw/d. The animals were treated 10 times during a period of 14 days. On the application area neither erythema nor edema of the skin were observed. The administration of 1000 mg/kg bw/d showed no toxicologically significant effects.

Positive control:

Not required for this study type

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least daily

DETAILED CLINICAL OBSERVATIONS: No

DERMAL IRRITATION: Yes
- Time schedule for examinations: at least daily
Erythema was scored using the Draize scale. The evaluation of swelling (in males and in females on days 0, 2, 6 ,9, 13, 16, 20, 23, 27) was done by measuring the skinfold thickness on the back in the center of the application area using a cutimeter or skinfold caliper. Measurements were taken at two different locations within the treatment area. From these a mean value was calculated

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Weekly

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
CBC Complete blood cell count
DIFF white blood cell differential
LEUCO Leucocytes
ERY Erythrocytes
HB Hemoglobin
HCT Hematocrit, calculated
MCV Mean Corpuscular Volume
MCH Mean Corpuscular Hemoglobin, calculated
MCHC Mean Corpuscular Hemoglobin Concentration, calculated
THRO Platelets/Thrombocytes
NEUTRO Neutrophils
LYMPH Lymphocytes
MONO Monocytes
EOS Eosinophils
BASO Basophils
ATYP Atypical Leucocytes

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Glucose tests were carried out on relative day 27. Laboratory tests on blood samples were carried out at the end of the 4-week treatment period (relative day 28). Blood samples for glucose determination were taken from fasted, non-anesthetized animals. The blood was obtained from the distal vessels of the tail. The blood for the other tests was withdrawn from animals under deep diethyl ether anesthesia by heart puncture at the time of necropsy.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

ORGAN WEIGHTS: Yes
The following exsanguinated organs of the animals sacrificed at necropsy were weighed in the unfixed state: adrenal glands, brain, epididymides, heart, kidneys, liver, spleen, testes, thymus. The organ weights are specified in both absolute and relative terms. The relative organ weights were calculated by normalization to 100 g body weight (individual organ weight divided by body weight times 100). The body weight used as a basis for this calculation was determined immediately prior to necropsy of the pertinent animal.

HISTOPATHOLOGY: Yes

Statistics:

The quantitative results for individual animals were used to calculate arithmetic group means and standard deviations. The results for the groups that received the test substance were compared with those for the control group and significant differences indicated by "+" for p<0.05 and "++" for p<0.01. In case of numbers of values too low to calculate test statistics this is indicated by "nc".
The Dunnett test was used for body weight, body weight gain, feed consumption and organ weight data (relative organ weights subsequent to logarithmic transformation)..

Clinical signs:

no effects observed

Description (incidence and severity):

No systemic clinical signs were observed

Dermal irritation:

no effects observed

Description (incidence and severity):

Skin reddening was not observed either in the control or in the dose group. During the entire treatment period, the mean skinfold thickness of the 1000 mg/kg group animals was comparable to the controls.

Mortality:

no mortality observed

Description (incidence):

No animal died throughout the entire treatment period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

In males and in females, the body weights and the body weight development were not impaired. Through the entire study, in females at 1000 mg/kg bw/d, the body weights were slightly higher than in the controls.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

In male and in female animals, the food consumption per animal was unaffected by treatment. In males, at day 7 a very slight decrease of the daily feed intake per kg body weight was calculated. This statistically significant effect is regarded as incidental.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There were no effects of treatment observed, neither on red blood cells, nor on leucocyte counts, on hemogram, or on blood coagulation. The statistically significantly decreased hemoglobin and hematocrit values in males are considered incidental. These effects were observed only in one sex, no individual values in the dose group are below the lower limit of the biological range for animals of this age. Additionally, the control values are very high, they exceed the 2s range.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no toxicologically significant changes in clinical chemical parameters. The observed statistically significant differences of the activity of alanine aminotransferase and of the concentration of triglycerides and creatinine in males, as well as of the concentration of inorganic phosphate in females are considered incidental. These effects were observed only in one sex, and all individual values are in the biological range for animals of this age.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No significant differences to the controls were observed.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no local or systemic histopathological findings attributable to the test substance in the organs/tissues examined.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Skin reactions

Details on results:

Regarding survival rate, state of health, or general behavior of the animals there was no difference between the treated and untreated animals. At 1000 mg/kg no macro- or microscopical skin findings were observed. At 1000 mg/kg the feed consumption and the body weights did not differ
lexicologically significantly from those in the controls. Hematology tests afforded no evidence for a treatment-related effect at 1000 mg/kg. Neither the hematopoetic system nor the red and white blood cell populations or blood coagulation were affected. The results of clinical laboratory tests, gross pathology, organ gravimetry and histopathology afforded no evidence for a treatment-related effect.

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Remarks:

Local effects

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Conclusions:

A 28-day dermal toxicity study in the rat was performed at the limit dose of 1000 mg/kg bw/d. A NOAEL of1000 mg/kg bw/d is reported for local and systemic effects.

Executive summary:

In this sub-acute repeated dose toxicity study, groups of 5 male and 5 female Wistar rats were administered spirodiclofen at dose levels of 0 (controls) and 1000 mg/kg bw/d by dermal application. Analytical determinations of stability and homogeneity were not performed because the test substance was applied undiluted and only moistened with water immediately before application. Males and females received 22 applications within a total period of 28 days. The  exposure time per day was 6 hours.  General observations: No effects on body weight development, food consumption, clinical signs, or skin reactions were seen.  Clinical chemistry and hematology did not reveal any treatment-related changes. The results of gross pathology, organ weight analysis and histopathology did not reveal evidence of a treatment-related effect.  In conclusion, neither systemic nor local skin effects were observed after a dermal administration of 1000 mg/kg bw/d to rats for four weeks.  1000 mg/kg bt/d is therefore regarded as the NOAEL for local and systemic effects in male and female rats.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Experimental exposure time per week (hours/week):

30

Species:

rat

Quality of whole database:

A GLP- and guideline compliant 28-day dermal toxicity study in the rat (Klimisch =1) is available

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

9 February 1998 to 4 May 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: 67/548/EEC

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Standard laboratory species/strain with background control data available

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 7-8 weeks
- Weight at study initiation: 223-250 g (M), 200-207 g (F)
- Fasting period before study: none
- Housing: individual
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least five days

DETAILS OF FOOD AND WATER QUALITY:
The rations consisted of fixed-formula standard diet (Altromin 1324 (= pelletized 1321) Diet for Rats and Mice) supplied by Altromin GmbH, and of tap water (drinking bottles). Feed and water were available ad libitum and were supplied in polycarbonate bottles and in stainless steel cage cover, respectively. The nutritive composition and the contaminant-content of the standard diet were routinely spot-checked and analyzed. The tap water complied with the German Drinking Water Ordinance.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-22
- Humidity (%): 50-60
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 18 February 1998 To: 17 March 1998

Type of coverage:

semiocclusive

Vehicle:

unchanged (no vehicle)

Remarks:

The test material was applied as a solid onto a wet gauze pad

Details on exposure:

Two days before the start of the treatment the back and flanks of the rats were shorn. Any regrowth of hair was shaved off the skin areas marked for treatment twice weekly. For each animal the required amount of test substance was weighed and transferred to the wet gauze-layer (5.5 x 5.5 cm = 30.25 cm2) of a sterile patch and placed on the rat's back. The gauze strip was secured in place using adhesive stretch tape (8 x 23 cm). Additionally, the mobility of the rats was impaired by a rat jacket. The gauze strip has the same size as the application area, i.e. 30.25 cm2. Thus, the application area was greater than 10% of the body surface area of the rat. After an exposure time of 6 hours the fixing bandage and the gauze strip were removed and the treated area cleaned with soap and water. Control animals were treated with tap water.

Analytical verification of doses or concentrations:

no

Remarks:

Analytical determinations of stability and homogeneity were not performed because the test substance was applied undiluted.

Duration of treatment / exposure:

The test material was applied for 6 hours/day

Frequency of treatment:

The test material was applied daily; 5 days/week for 28 days (total 22 applications). On the first three weeks of the study, the formulations were applied on 5 days per week, and in the fourth week also on Saturday and Sunday. Thus, both males and females were dosed daily over the final seven days prior to terminal sacrifice.

Dose / conc.:

0 mg/kg bw/day

Remarks:

Control; water only

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Test material moistened with water

No. of animals per sex per dose:

Groups of 5 male and 5 female rats per dose.

Control animals:

yes, concurrent no treatment

Details on study design:

Groups of 5 male and 5 female Wistar rats of the strain Hsd Cpb: WU were administered spirodiclofen, at levels of 0 and 1000 mg/kg bw/d by dermal application. Analytical determinations of stability and homogeneity were not performed because the test substance was applied undiluted and only moistened with water immediately before application. Males and females received 22 applications within a total period of 28 days. The exposure time per day was 6 hours. The doses were based on the results of a dose range finding study in female rats. In that study, the doses were 0 and 1000 mg/kg bw/d. The animals were treated 10 times during a period of 14 days. On the application area neither erythema nor edema of the skin were observed. The administration of 1000 mg/kg bw/d showed no toxicologically significant effects.

Positive control:

Not required for this study type

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least daily

DETAILED CLINICAL OBSERVATIONS: No

DERMAL IRRITATION: Yes
- Time schedule for examinations: at least daily
Erythema was scored using the Draize scale. The evaluation of swelling (in males and in females on days 0, 2, 6 ,9, 13, 16, 20, 23, 27) was done by measuring the skinfold thickness on the back in the center of the application area using a cutimeter or skinfold caliper. Measurements were taken at two different locations within the treatment area. From these a mean value was calculated

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Weekly

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
CBC Complete blood cell count
DIFF white blood cell differential
LEUCO Leucocytes
ERY Erythrocytes
HB Hemoglobin
HCT Hematocrit, calculated
MCV Mean Corpuscular Volume
MCH Mean Corpuscular Hemoglobin, calculated
MCHC Mean Corpuscular Hemoglobin Concentration, calculated
THRO Platelets/Thrombocytes
NEUTRO Neutrophils
LYMPH Lymphocytes
MONO Monocytes
EOS Eosinophils
BASO Basophils
ATYP Atypical Leucocytes

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Glucose tests were carried out on relative day 27. Laboratory tests on blood samples were carried out at the end of the 4-week treatment period (relative day 28). Blood samples for glucose determination were taken from fasted, non-anesthetized animals. The blood was obtained from the distal vessels of the tail. The blood for the other tests was withdrawn from animals under deep diethyl ether anesthesia by heart puncture at the time of necropsy.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

ORGAN WEIGHTS: Yes
The following exsanguinated organs of the animals sacrificed at necropsy were weighed in the unfixed state: adrenal glands, brain, epididymides, heart, kidneys, liver, spleen, testes, thymus. The organ weights are specified in both absolute and relative terms. The relative organ weights were calculated by normalization to 100 g body weight (individual organ weight divided by body weight times 100). The body weight used as a basis for this calculation was determined immediately prior to necropsy of the pertinent animal.

HISTOPATHOLOGY: Yes

Statistics:

The quantitative results for individual animals were used to calculate arithmetic group means and standard deviations. The results for the groups that received the test substance were compared with those for the control group and significant differences indicated by "+" for p<0.05 and "++" for p<0.01. In case of numbers of values too low to calculate test statistics this is indicated by "nc".
The Dunnett test was used for body weight, body weight gain, feed consumption and organ weight data (relative organ weights subsequent to logarithmic transformation)..

Clinical signs:

no effects observed

Description (incidence and severity):

No systemic clinical signs were observed

Dermal irritation:

no effects observed

Description (incidence and severity):

Skin reddening was not observed either in the control or in the dose group. During the entire treatment period, the mean skinfold thickness of the 1000 mg/kg group animals was comparable to the controls.

Mortality:

no mortality observed

Description (incidence):

No animal died throughout the entire treatment period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

In males and in females, the body weights and the body weight development were not impaired. Through the entire study, in females at 1000 mg/kg bw/d, the body weights were slightly higher than in the controls.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

In male and in female animals, the food consumption per animal was unaffected by treatment. In males, at day 7 a very slight decrease of the daily feed intake per kg body weight was calculated. This statistically significant effect is regarded as incidental.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There were no effects of treatment observed, neither on red blood cells, nor on leucocyte counts, on hemogram, or on blood coagulation. The statistically significantly decreased hemoglobin and hematocrit values in males are considered incidental. These effects were observed only in one sex, no individual values in the dose group are below the lower limit of the biological range for animals of this age. Additionally, the control values are very high, they exceed the 2s range.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no toxicologically significant changes in clinical chemical parameters. The observed statistically significant differences of the activity of alanine aminotransferase and of the concentration of triglycerides and creatinine in males, as well as of the concentration of inorganic phosphate in females are considered incidental. These effects were observed only in one sex, and all individual values are in the biological range for animals of this age.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No significant differences to the controls were observed.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no local or systemic histopathological findings attributable to the test substance in the organs/tissues examined.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Skin reactions

Details on results:

Regarding survival rate, state of health, or general behavior of the animals there was no difference between the treated and untreated animals. At 1000 mg/kg no macro- or microscopical skin findings were observed. At 1000 mg/kg the feed consumption and the body weights did not differ
lexicologically significantly from those in the controls. Hematology tests afforded no evidence for a treatment-related effect at 1000 mg/kg. Neither the hematopoetic system nor the red and white blood cell populations or blood coagulation were affected. The results of clinical laboratory tests, gross pathology, organ gravimetry and histopathology afforded no evidence for a treatment-related effect.

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Remarks:

Local effects

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Conclusions:

A 28-day dermal toxicity study in the rat was performed at the limit dose of 1000 mg/kg bw/d. A NOAEL of1000 mg/kg bw/d is reported for local and systemic effects.

Executive summary:

In this sub-acute repeated dose toxicity study, groups of 5 male and 5 female Wistar rats were administered spirodiclofen at dose levels of 0 (controls) and 1000 mg/kg bw/d by dermal application. Analytical determinations of stability and homogeneity were not performed because the test substance was applied undiluted and only moistened with water immediately before application. Males and females received 22 applications within a total period of 28 days. The  exposure time per day was 6 hours.  General observations: No effects on body weight development, food consumption, clinical signs, or skin reactions were seen.  Clinical chemistry and hematology did not reveal any treatment-related changes. The results of gross pathology, organ weight analysis and histopathology did not reveal evidence of a treatment-related effect.  In conclusion, neither systemic nor local skin effects were observed after a dermal administration of 1000 mg/kg bw/d to rats for four weeks.  1000 mg/kg bt/d is therefore regarded as the NOAEL for local and systemic effects in male and female rats.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

1 000

Study duration:

subacute

Species:

rat

Quality of whole database:

A GLP- and guideline compliant 28-day dermal toxicity study in the rat (Klimisch =1) is available

Justification for classification or non-classification

Spirodiclofen has a harmonised classification for STOT RE 2 on the basis of the available data.   The relevant part of the RAC Conclusion is summarised below:

Mouse studies

RAC concluded that in the oral repeated dose toxicity studies in the mouse (13-week & 18-month) there was no evidence for severe liver effects such as organ lesions or dysfunction. Classification for STOT RE based on the mouse studies was not justified.

Rat studies

The effect levels in the 4-week, 14-week and 2-year rat repeated dose toxicity studies were mostly above the upper limit for STOT RE classification.  Increased small cortical vacuolisation of the adrenals (with increased severity grading) was observed in males at dose levels of 6.6 and 32.1 mg/kg bw/d and higher in the 14-week study (below the guidance valuefor STOT RE 2 classification).  However this was within the range of historical controls.  Additionally, the results from the combined chronic toxicity/carcinogenicity, 2-generation reproductive toxicity, neurotoxicity and immunotoxicity studies did not warrant classification for STOT RE.

Dog studies
In the available repeated dose toxicity studies in dogs (4-week, 8-week, 14-weekand 1-year) many parameters of various organ systems were affected including the haematological system, the liver and the adrenals. The observed adrenal effects in dogs (cytoplasmic vacuolisation and mononuclear cell infiltration adrenal cortex effects) were considered not to be severe and not to fulfil the CLP criteria for STOT RE classification.  Effects on the haematological system were not observed in the 8-week and 1-year dog studies. However, they were seen in the 4-week and 14-week dog studies.  In both studies the effect levels were below the upper limit for STOT RE 2.  In the 4-week study reduced erythrocytes, Hb and Hct were observed. In the 14-week study though, a dose related effect on Hb and Hct  evels and erythrocyte count was seen and at the highest dose level a 20% decline of these parameters was observed which is considered a consistent and adverse effect.  The liver was also identified as a target organ in dogs. Effects included increased organ weight and increased biochemical parameters. Also hepatocellular necrosis was observed although at effect-levels below the upper limit for STOT RE 2 classification.

STOT RE 2 classification is therefore based on haematology and liver effects and apply to all routes of exposure.