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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1998-07-21 - 2000-01-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-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 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
3-methylpyrazole
EC Number:
215-925-7
EC Name:
3-methylpyrazole
Cas Number:
1453-58-3
Molecular formula:
C4H6N2
IUPAC Name:
3-methyl-1H-pyrazole
Test material form:
other: liquid

Test animals

Species:
mouse
Strain:
B6C3F1
Sex:
male/female

Administration / exposure

Route of administration:
oral: drinking water
Vehicle:
water
Details on oral exposure:
To achieve the target dose levels the test article concentrations in the water were adjusted weekly according to mean water consumption and mean body weight of the animals.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples for the determination of the concentration, homogeneity and stability of the test article mixtures were drawn from the first test article preparations. Intercurrent samples for the determination of the concentration were drawn twice during the study, at monthly intervals. All samples were stored at room temperature prior to analysis by RCC Ltd, Environmental Chemistry and Pharmanalytics Division, CH-4452 Itingen by HPLC.
Duration of treatment / exposure:
13 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
5 mg/kg bw (total dose)
Remarks:
Doses / Concentrations:
5 mg/kg
Basis:
nominal in water
Dose / conc.:
10 mg/kg bw (total dose)
Remarks:
Doses / Concentrations:
10 mg/kg
Basis:
nominal in water
Dose / conc.:
20 mg/kg bw (total dose)
Remarks:
Doses / Concentrations:
20 mg/kg
Basis:
nominal in water
Dose / conc.:
40 mg/kg bw (total dose)
Remarks:
Doses / Concentrations:
40 mg/kg
Basis:
nominal in water
No. of animals per sex per dose:
10 animals per sex and dose group
Control animals:
yes, concurrent no treatment
Positive control:
no

Examinations

Observations and examinations performed and frequency:
Observations:

Clinical signs: At least once daily

Viability/mortality: Twice daily

Food consumption: Weekly

Water consumption: Weekly

Body weights: Weekly

Ophthalmoscopic examinations: Pretest: on all mice per sex and group.
At week 13: control and high dose animals.
At week 17: control and high dose animals.
After the application of a mydriatic solution (CIBA Vision AG, 3172 Niederwangen / Switzerland) the cornea, lens, anterior chamber, vitreous body and ocular fundus of both eyes were examined using a Heine Miroflex 2 Ophthalmoscope (Eisenhut Vet AG, Allschwil / Switzerland) and the results were recorded. Unless otherwise indicated in the table, the contralateral eye was without abnormalities.

Sacrifice and pathology:
All animals were weighed and necropsied. Descriptions of all macroscopic abnormalities were recorded. Necropsies were performed by experienced prosectors supervised by a veterinary pathologist. All animals surviving to the end of the observation period and all moribund animals will be anesthetized by intraperitoneal injection of sodium pentobarbitone and killed by exsanguination.

Samples of the following tissues and organs will be collected from all animals at necropsy and fixed in neutral phosphate buffered 4% formaldehyde solution:
Adrenal glands

Aorta

(Auricles)

Bone marrow (femur)

Brain - including section of medulla/pons,

cerebral and cerebellar cortex

Cecum

Colon

Duodenum

Epididymides

Esophagus

(Exorbital lacrymal glands)

Eyes with optic nerve and Harderian gland

Femur - including articular surface

Heart

Ileum

Jejunum

Kidneys

Liver with gall bladder Lungs, infused with formalin Lymph nodes - mesenteric, mandibular Mammary gland area
Nasal cavity Ovaries Pancreas Pituitary gland Prostate gland Rectum

Salivary glands - mandibular, sublingual

Sciatic nerve

Seminal vesicles

Skeletal muscle

Skin

Spinal cord - cervical, midthoracic, lumbar Spleen

Sternum with bone marrow

Stomach

Testes

Thymus

Thyroid gland / parathyroid gland Trachea

Urinary bladder, infused with formalin

Uterus

Vagina

All gross lesions
Other examinations:
Blood samples for hematology and clinical biochemistry were collected from all allocation A animals at 13 weeks and from all allocation B animals at 17 weeks under light ether anesthesia. The animals were fasted for approximately 18 hours before blood sampling but allowed access to water ad libitum. Blood samples were collected early in the working day between the hours of 06.00 and 08.35 to reduce biological variation caused by circadian rhythms. Blood samples were drawn from the retro-orbital plexus using a micro-hematocrit glass capillary tube.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality 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):
effects observed, treatment-related
Description (incidence and severity):
Water consumption over treatment was marginally increased in females of groups 4 (20 mg/kg) and 5 (40 mg/kg) and slightly increased in males of groups 2 (5 mg/kg), 3 (10 mg/kg), 4 (20 mg/kg) and 5 (40 mg/kg).
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The Clara cell alteration noted in mice of groups 10, 20, and 40 mg/kg following the treatment period is considered to represent a mixed degenerative and regenerative process indicating a probable systemic effect of the test article.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: toxicological endpoint: effects on Clara cells (lung)

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
10 mg/kg bw (total dose)
System:
respiratory system: lower respiratory tract
Organ:
lungs
Treatment related:
yes

Applicant's summary and conclusion

Conclusions:
Based on these results the "no-observed-adverse-effect level (NOAEL)" in this study is considered to be the target dose of 5 mg/kg body weight/day corresponding to a test article intake of 5.20 mg/kg body weight/day in male mice and 5.31 mg/kg body weight/day in female mice.

The dose of 10 mg/kg/day is considered to represent a threshold dose level for the Clara cell alteration, since the incidence and severity of this lesion were substantially lower at this dose level when compared with the higher doses.
Executive summary:

Treatment of SPF-bred Crl:B6C3Fl mice with MP (3-Methylpyrazole) via their drinking water for a period of 13 weeks at doses of 5, 10, 20 or 40 mg/kg had no effect on survival, clinical signs, food consumption, body weight, ophthalmoscopy findings, hematology and clinical biochemistry parameters, organ weights or macroscopical findings. The increased water consumption noted in females of groups 4 (20 mg/kg) and 5 (40 mg/kg) as well in all male groups treated with MP reversed during the 4-week treatment-free recovery period and might reflect impaired palatability of the water by the test article resulting in undetected water spillage. The only other findings observed in this study were confined to histopathology. The Clara cell alteration noted in mice of groups 3 (10 mg/kg), 4 (20 mg/kg), and 5 (40 mg/kg) following the treatment period is considered to represent a mixed degenerative and regenerative process indicating a probable systemic effect of the test article. This Clara cell alteration was dose-dependent in incidence and severity in the groups affected. At the end of the treatment-free recovery period, Clara cell regeneration and repair were noted in association with a persistent Clara cell alteration. The 4-week treatment-free recovery period was insufficient to allow for full reversal of this Clara cell alteration. However the incidence and mean severity of this alteration showed a dose-related decrease in the recovery mice when compared with the mice sacrificed at termination of the treatment period. No Clara cell alterations were noted in mice of group 2 (5 mg/kg). Based on these results the "no-observed-adverse-effect level (NOAEL)" in this study is considered to be the target dose of 5 mg/kg body weight/day corresponding to a test article intake of 5.20 mg/kg body weight/day in male mice and 5.31 mg/kg body weight/day in female mice. The dose of 10 mg/kg/day is considered to represent a threshold dose level for the Clara cell alteration, since the incidence and severity of this lesion were substantially lower at this dose level when compared with the higher doses.

Based on the publications of the German MAK commission for napthalene ( see http://onlinelibrary.wiley.com/doi/10.1002/3527600418.mb9120d0021/full) and of the German Federal Environmental Agency (see http://www.umweltbundesamt.de/gesundheit/publikationen/ad-hoc/Naphthalin.pdf) clara cells of mice are considered to have a specific sensitivity to lung toxicants, because they have a cytochrome P450 dependent oxidation potency, which is 1 to 2 magnitudes higher as of humans (see also Buckpitt A. et al., Relationship of cytochrome P450 activity to Clara cell cytotoxicity. IV. Metabolism of naphthalene and naphthalene oxide in microdissected airways from mice, rats, and hamsters. , http://www.ncbi.nlm.nih.gov/pubmed/7838135). This means, that effects on clara cells of mice are not transferable to man a priori.