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Diss Factsheets

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report date:
1999

Materials and methods

Test guidelineopen allclose all
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

Test material

Constituent 1
Reference substance name:
3-(3,5-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-(3,5-Dichlorophenyl)-2-oxo-1-oxaspiro[4.5]dec-3-en-4-yl 2,2-dimethylbutanoate

Test animals

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

Administration / exposure

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.
Doses / concentrationsopen allclose all
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

Examinations

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)..

Results and discussion

Results of examinations

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.

Effect levels

open allclose all
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

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

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.