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Diss Factsheets

Administrative data

Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 Apr 1999 - 13 Oct 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report date:
2000

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Version / remarks:
adopted 1981
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
433-460-1
EC Name:
-
Cas Number:
210880-92-5
Molecular formula:
C6H8ClN5O2S
IUPAC Name:
(E)-N'-[(2-chloro-1,3-thiazol-5-yl)methyl]-N-methyl-N''-nitroguanidine

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The rat is a common species for toxicological testing and recommended by the guideline.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., North Carolina, USA
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approximately 8 weeks
- Weight at study initiation: 240 - 317 g (males), 171 - 213 g (females)
- Fasting period before study: not applicable
- Housing: individually in suspended, stainless steel cages
- Diet: Certified rodent diet (#8728C, Harlan Teklad), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 16 days, collared for 18 h/day for 5 days before treatment

DETAILS OF FOOD AND WATER QUALITY: Diet and water was routinely checked, no signs of contaminants were found

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 26
- Humidity (%): 30 - 70
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 29 Apr To: 28 May 1999

Administration / exposure

Type of coverage:
occlusive
Vehicle:
water
Details on exposure:
TEST SITE
- Area of exposure: 5x5 cm
- % coverage: 10
- Type of wrap if used: gauze pad, fixed with surgical micropore tape and wrapped with Vetwrap®.
- Time intervals for shavings or clipplings: one week prior to initial treatment, one day prior to first treatment and whenever neccessary thereafter

REMOVAL OF TEST SUBSTANCE
- Washing (if done): sites were wiped with water-moistened paper tissues/towels
- Time after start of exposure: 6h (when exposure was terminated)

TEST MATERIAL
- Amount applied (volume or weight with unit): weight was based on the body weight of the test animal
- Constant volume or concentration used: no
- For solids, paste formed: no, but application site was moistenend with water

VEHICLE
- Justification for use: water
- Amount(s) applied (volume or weight with unit): 1 mL + water for moisturizing application site

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes (flexible plastic collars)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test material was confirmed using HPLC. It was verified that TI-435 was stable for at least 53 weeks when stored in a refrigerator set to maintain 2 to 8 °C.
Duration of treatment / exposure:
6 h/day for 29 days
Frequency of treatment:
7 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the low dose was chosen to cause no toxicity, the mid dose should cause an intermediate level of toxicity and in the high dose toxic effects should be observed but no severe skin effects or mortality meaningful evaluation
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- parameters checked: twice daily: mortality and moribundity, signs of poor health or abnormal behavior, once daily: indications of a toxic effect

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly
- parameters observed included changes in skin, fur, eyes, and mucous membranes; occurrences of secretions and excretions; and autonomic activity (e.g., lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in posture and reactivity to handling and the presence of clonic or tonic movements, stereotypies (e.g., excessive grooming, circling) or bizarre behavior (e.g., self-mutilation, walking backwards) and gait abnormalities.

DERMAL IRRITATION: Yes
- Time schedule for examinations: scored daily (modified Draize method)

BODY WEIGHT: Yes
- Time schedule for examinations: one day before treatment (-Day 1), on Day of first treatment (Day 1), and on Days 7, 14, 21, 28, and 30 (day of necropsy)

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: Yes

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before initiation of treatment and during Week 4
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 5 (at termination)
- Anaesthetic used for blood collection: Yes (sodium pentobarbital)
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters checked: red blood cell (erythrocyte) count, hemoglobin, hematocrit, mean cell volume, mean cell hemoglobin, mean cell hemoglobin concentration, platelet count, white blood cell (leukocyte) count, differential blood cell count (segmented neutrophil count, lymphocyte count, monocyte count, eosinophil count, basophil count), blood cell morphology, reticulocyte count, activated partial thromboplastin time and prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 5 (at termination)
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters checked:
glucose, urea nitrogen, creatinine, total protein, albumin, globulin, albumin/globulin ratio, total cholesterol, total bilirubin, alkaline phosphatase, alanine aminotransferase, gamma glutamyltransferase, aspartate aminotransferase, calcium, inorganic phosphorus, magnesium, sodium, potassium, chloride, creatine kinase and sorbitol dehydrogenase

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once during Week 4 prior to dosing
- Dose groups that were examined: all
- Battery of functions tested: expanded clinical observation during handling and in an open field and for sensory activity, grip strength and motor activity.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All surviving animals were anesthetized with sodium pentobarbital after blood collection and sacrificed. Necropsy included a macroscopic examination of the external features of the carcass; external body orifices; the abdominal, thoracic, and cranial cavities; and organs/tissues.
Organ weights were recorded for adrenals, brain, epididymis, kidney, heart, liver, ovaries, spleen, testis, thymus and uterus.

HISTOPATHOLOGY: Yes
- Tissues were collected and preserved in 10% formalin with the following deviations:
* Fixed in Davidson's, ** preserved lobes were perfused with 10% neutral-buffered formalin, *** Fixed in Bouin's, subsequently rinsed with tap water, and transferred to 70% alcohol.
- Tissues collected: adrenal, aorta (thoracic), bone [femur (articular surface of the distal end) and sternum], bone marrow (femur and sternum), brain, cecum, colon, duodenum, epididymis, esophagus, eyes* (2, retina, optic nerve), gross lesions, heart, ileum, jejunum, kidneys, larynx, liver, lung**, mammary gland (female), lymph node (mesenteric), nasal turbinates***, ovaries, pancreas, pharynx***, pituitary, prostate, rectum, salivary gland (mandibular), sciatic nerve, seminal vesicle, skin (treated dorsal, untreated dorsal and untreated ventral inguinal), spinal cord (cervical, thoracic, and lumbar), spleen, stomach, testis***, thigh musculature thymus throidy (with parathyroid), trachea, urinary bladder and uterus
- embedding media: paraffin
- staining: hematoxylin and eosin
- animals: control and high-dose groups and from each animal that died or was sacrificed unscheduled, macroscopic lesions and skin (treated and untreated) were also examined microscopically from each animal in the low- and mid-dose groups.
Statistics:
Homogenity of variance was checked with Levene's test. If heterogenity of of variance at p < 0.05, transformations were used to stabilize the variance. Comparison tests took variance of heterogeneity into consideration.

To analyze grip strength, motor activity, body weights, body weight changes, food consumption, food efficiency, continuous clinical pathology values, and organ weight data, an One-way analysis of variance, ANOVA, was used. If a significant difference was found, Dunnett's t-test was used for control versus treated group comparisons.

Significance was set at p ≤ 0.05

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
There was no difference observed between control and treatment animals. That also included the expanded clinical observations during neurobehavioral testing.
Dermal irritation:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test material-related dermal observations noted. A single incidence of very slight edema was noted at the application site in a female given 300 mg/kg bw/day. However, since it was a sporadic finding and no dose-response was found, the observation was considered incidental.

Summarized data can be found in Attachment 1 in the attached background material.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female receiving 100 mg/kg bw/day and one female receiving 1000 mg/kg bw/day died prematurely (Day 15). In both cases, cause of death was not determinable but no treatment-related signs were found and the deaths were therefore considered incidental.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Only incidental differences in body weight and body weight gains were observed. The only finding was reduced body weight changes in males treated with 1000 mg/kg bw/day in the first week (-61%) but this was not seen in any other week. However, the lower body weight gain in the firt week resulted in overall lower body weight gain in males in this treatment group throughout the study (-20% for Day 1 to 28). Sice this was due to the finding in Week 1, it was also considered incidental. In females, no differences were found for body weight and body weight gain.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Sporadical differences in food consumptions were found for both sexes at the mid and high-dose treatment group. However, the differences ranged from -10.1 to +11% and no dose- or time-dependency could be established. they were therefore considered incidental.
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
Corresponding to the reduced body weight gain seen in week 1 for males (1000 mg/kg bw/day), the food efficiency was also reduced. As for the finding in body weight change it was considered incidental. Additionally, food efficiency in the 300 mg/kg bw/day treatment group (males) was reduced in Week 2 but this was the only interval in which this was observed and it was therefore considered incidental.
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
not applicable
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no differences between control and treatment groups.
Haematological findings:
no effects observed
Description (incidence and severity):
There were no differences between control and treatment groups.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no differences between control and treatment groups.
Endocrine findings:
not examined
Description (incidence and severity):
not applicable
Urinalysis findings:
not examined
Description (incidence and severity):
not applicable
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no differences between control and treatment groups.
Immunological findings:
not examined
Description (incidence and severity):
not applicable
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no differences between control and treatment groups.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no differences between control and treatment groups.
Neuropathological findings:
no effects observed
Description (incidence and severity):
There were no differences between control and treatment groups.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no differences between control and treatment groups.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
There were no differences between control and treatment groups.

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 effects observed

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
The effect of repeated dermal application of the test substance was analyzed in a 28 day study in rats under OECD guideline 410 and GLP. Under the conditions of the test, no toxicity was observed after repeated dermal treatment with the test substance, so that the NOAEL was set at 1000 mg/kg bw, the highest concentration tested.
The study was done according to OECD guideline 410 and under GLP.