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Administrative data

Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29. Jun - 22. Jul 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report date:
1990

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Version / remarks:
1981
Deviations:
yes
Remarks:
methodological deficiencies (treatment time was shorter (15 days))
Qualifier:
according to guideline
Guideline:
EPA OPP 82-2 (Repeated Dose Dermal Toxicity -21/28 Days)
Version / remarks:
1982
Deviations:
no
GLP compliance:
yes
Limit test:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
428-040-8
EC Name:
-
Cas Number:
138261-41-3
Molecular formula:
C9H10ClN5O2
IUPAC Name:
2-chloro-5-{[2-(nitroimino)imidazolidin-1-yl]methyl}pyridine

Test animals

Species:
rabbit
Strain:
New Zealand White
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Interfauna, UK Limited, Huntingdon, England
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 10 - 13 weeks
- Weight at study initiation: 2.65 - 3.43 kg (females), 2.93 - 3.09 kg (males)
- Housing: individually in Cellidor Rabbit cages with bedding (low-dust wood granulate from Ssniff Spezialdiaten GmbH, Soest)
- Diet: "ssniff K Alleindiät für Kaninchen" (Ssniff Spezialdiäten GmbH, Soest/Westfalen, Germany), given daily with automatic rabbit feeders
- Water: tap water, ad libitum
- Acclimation period: 15 days

DETAILS OF FOOD AND WATER QUALITY: The water was checked regularly, no influence on the objective of the study was indicated

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): approx. 50%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 29. Jun To: 22. Jul 1988

Administration / exposure

Type of coverage:
semiocclusive
Vehicle:
other: 2% (v/v) Cremophor EL in physiological saline solution
Details on exposure:
TEST SITE
- Area of exposure: Backs and flanks
- % coverage: > 10
- Type of wrap if used: the test article was covered with a muslin cloth, that was fixed with adhesive tape (Fixomullstretch: Beiersdorf AG, Hamburg)
- Time intervals for shavings or clipplings: one day before starting treatment, then twice weekly

REMOVAL OF TEST SUBSTANCE
- Washing: soap and water
- Time after start of exposure: 6 h

TEST MATERIAL
- Amount applied: 1000 mg/kg bw/day
- Concentration: 40%
- Constant volume or concentration used: yes
- For solids, paste formed: yes

VEHICLE
- Justification for use and choice of vehicle: not specified
- Amount applied: 1.5 mL/kg bw formulation agent
- Concentration (if solution): 2% (v/v) Cremophor EL in physilogical saline solution

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test article in the application formulation was confirmed analytically for 0 and 24 h, resulting in 95.4% and 95.7% of the nominal dose for 0 and 24 h, respectively.
Duration of treatment / exposure:
15 days
Frequency of treatment:
5 days/week
Doses / concentrations
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5
Control animals:
yes
Details on study design:
- Dose selection rationale: The dose was set on the basis of the results of a preliminary study performed on the rabbit (study no: T4027690)
- Fasting period before blood sampling for clinical biochemistry: not reported
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily

DETAILED CLINICAL OBSERVATIONS: No data

DERMAL IRRITATION: Yes
- Time schedule for examinations: daily

BODY WEIGHT: Yes
- Time schedule for examinations: prior to start of the experiment and weekly thereafter (on Days 0, 7, 14, 22 and 23)

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: before commencement and after the three-week treatment.
- Anaesthetic used for blood collection: no data
- Animals fasted: not reported
- How many animals: all animals
- Parameters checked: erythrocyte and leucocyte count, hemoglobin, MCV (calculation of mean corpuscular volume of individual erythrocytes), hematocrit, thrombocyte count, MCH (mean hemoglobin content of individual erythrocytes), MCHC (mean hemoglobin concentration of erythrocytes), differential blood count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: as for hematology
- Animals fasted: no data
- How many animals: all animals
- Parameters checked: Aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, urea, glucose, creatinine, bilirubin, total protein, cholesterin, inorganic phosphate, sodium, potassium, calcium, chloride;
- Time schedule for collection of liver tissue: after necropsy
- How many animals: all animals
- Parameters checked: N-demethylase, O-demethylase, cytochrome P-450, triglycerides

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Organs fixed in Bouin's fluid: treated and untreated skin, thyroids, lung, heart, liver, spleen, kidneys, adrenals, testes, epididymides, ovaries, uterus, sternum
- Organs fixed in formol calcium: liver, kidney
- absolute and relative organ weights recorded for: brain, thyroids, heart, lung, liver, kidneys, adrenals, spleen, testes, ovaries

HISTOPATHOLOGY: Yes
- Organs checked: treated and untreated skin, thyroids, lung, heart, liver, spleen, kidneys, adrenals, testes, epididymides, ovaries, uterus, sternum
Statistics:
Mean and standard deviation were calculated for food consumption, body weights, and for hematological observations and clinical chemistry. Groups were compared using the two-tailed U Test according to Mann and Whitney and Wilcoxon. Significance was defined as p < 0.05

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Soft feces was observed on Day 7 in one male animal of the control group and reduced food intake, stiff head position, transfixed bulging eyes, open mouth, salivation and wet nose in one female of the control group (caused by illness)
Dermal irritation:
no effects observed
Description (incidence and severity):
Redness and skin fold measurements did not differ for control and treated animals. Summarized data can be found in Attachment 1 and 2 in the attached background material.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Control: One female was sacrificed (Day 3) because of a fractured femur (the female was replaced)
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The body weights of the treated animals did not significantly differ from those of the control group.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Increased food consumption in treated males vs control animals in the first week (3.7%, statistically significant) were observed. Further, reduced food consumption in one control female on Day 14 caused by an illnes was evident.
Food efficiency:
not examined
Description (incidence and severity):
not applicable
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
not applicable
Ophthalmological findings:
not examined
Description (incidence and severity):
not applicable
Haematological findings:
no effects observed
Description (incidence and severity):
not applicable
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
not applicable
Endocrine findings:
not examined
Description (incidence and severity):
not applicable
Urinalysis findings:
not examined
Description (incidence and severity):
not applicable
Behaviour (functional findings):
not examined
Description (incidence and severity):
not applicable
Immunological findings:
not examined
Description (incidence and severity):
not applicable
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
- 1000 mg/kg bw: absolute and relative brain weight increased in males (7.3 and 6.8%, respectively), reduced absolute ovary weight (females, 35.3%), all compared to controls

Since these finidngs were slight, they were considered incidental.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No difference was found between the control and the treatment group.
Neuropathological findings:
not examined
Description (incidence and severity):
not applicable
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
not applicable
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
not applicable
Other effects:
not examined
Description (incidence and severity):
not applicable

Effect levels

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: no adverse effects observed up to and including this dose (highest dose tested)

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
No dermal irritation or any sign of toxicity was observed in this repeated dose study over 15 days with 1000 mg/kg bw/day of the test substance applied to the skin of rabbits. Accordingly, the NOAEL is set at 1000 mg/kg bw/day. The study was conducted according to OECD Guideline 410 but the application duration was shorter than suggested (15 days instead of at least 21 days). Therefore, the study is considered reliable with restrictions. However, as no signs indicating a toxic effect were observed, the NOAEL is acceptable.