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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP test according to guideline. The test report from the Japanese Ministry of Health & Welfare is not available, but all the data were validated by the Japanese authorities within the OECD SIDS program Information in this record is from therefore from the SIDS dossier, a reliable secondary source, and used as supportive information. Reliability assessment based on that given in SIDS dossier.

Data source

Referenceopen allclose all

Reference Type:
secondary source
Title:
Unnamed
Year:
2000
Reference Type:
publication
Title:
SIDS Initial Assessment Report for SIAM 14, Paris 26-28 March 2002.
Author:
Japan Ministry of Foreign Affairs, Economic Affairs Bureau, Second International Organizations division
Year:
2002
Bibliographic source:
OECD, Paris. http://www.chem.unep.ch/irptc/sids/OECDSIDS/16219753.pdf

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Principles of method if other than guideline:
No further information available.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Test substance: 5-Ethylidene-2-norbornene (CAS No. 16219-75-3)
- Source: Nippon Petrochemicals Co., Ltd, Lot No.6J01
- Purity: 99.4%
- Stability during use confirmed by gas chromatography.

Test animals

Species:
rat
Strain:
other: CrjCD (SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
Individual rats were identified by ear tattoo and cage cards.
- Age at study initiation: 5 weeks
- Diet (e.g. ad libitum): rat chow diet was provided ad libitum. 
- Water (e.g. ad libitum): Water was provided ad libitum
- Housing: Rats were housed individually in suspended wire mesh metallic cages

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-24 0C
- Humidity (%): 31-79%
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12 hour light-dark cycle.  

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
Doses of test article were administered by gavage in corn oil once/day at 5.0 ml/kg. 
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
28 days
Frequency of treatment:
once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 4, 20, 100 (mg/kg)
Basis:

No. of animals per sex per dose:
14 M, 14 F control and high dose, 7 M, 7 F mid and low dose
Control animals:
yes, concurrent vehicle
Details on study design:
Post-exposure period: 14 days

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Observations for mortality, morbidity and clinical signs were made at least once a day.

BODY WEIGHT: Yes
- Time schedule for examinations: Body wt  was determined at initiation and on dosing days 2, 5, 7, 10, 14, 21, and 28 and recovery days 2, 5, 7, 14 and at necropsy. 

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption was determined at initiation and on dosing days 2, 5, 7, 10, 14, 21, and 28 and recovery days 2, 5, 7, 14 and at necropsy. 

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water consumption was determined and unfasted urines were collected during dosing days 25-26 and recovery days 11-12. 

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Hematological analysis  was conducted on day 29 and on day 15 of recovery.  
- Parameters checked: Parameters examined were RBC, Hct, Hb, MCV, MCH, MCHC, platelets, reticulocytes, three coagulation factors, WBC and differential counts. 

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Hematological analysis and clinical chemistries were conducted on day 29 and on day 15 of recovery.  
- Parameters checked: GOT, GPT, Al-P, LDH, gamma-GTP, glucose, total cholesterol, triglycerides, bilirubin, BUN, creatinine, sodium, potassium, chloride, calcium, inorganic phosphate, total protein, albumin, and protein fractionation.

URINALYSIS: Yes
- Parameters checked: Urine was analyzed for pH, protein, glucose, ketones, urobilinogen, bilirubin, occult blood, color, sediment, volume, specific gravity, sodium, potassium, chloride, calcium, and inorganic phosphates.
Sacrifice and pathology:
At end of dosing and on day 15 of recovery, rats were sacrificed

GROSS PATHOLOGY: Yes, Brain, pituitary, thymus, thyroid, parathyroid, lung, heart, liver, kidneys, spleen, adrenal glands, testes and ovaries were weighed before fixation.
HISTOPATHOLOGY: Yes, 46 tissues/organs were fixed and prepared for histopathologic examination: liver, kidneys, spleen, heart, lung, brain, pituitary, adrenal glands, thyroid, parathyroid, thymus, mesenteric lymph nodes, pancreas, tongue, mandibular lymph nodes, submandibular gland, sublingual gland, parotid gland, zymbal gland, skin, sternum, femur, spine, skeletal muscle, thoracic aorta, pharynx, trachea, bronchi, esophagus, stomach, duodenum, jejunum, ileum, caecum, colon, rectum, bladder, seminal vesicle, prostate, ovary, uterus, vagina, ischiatic nerve, testes, epididymides, eye and Hardarian gland.
Statistics:
Bartlett's test and analysis of variance as necessary and Dunnett's test.  Kruskal-Wallis and Mann-Whitney U-test for histopathology.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no deaths attributable to test article administration.

BODY WEIGHT AND WEIGHT GAIN
At dosage termination, mean body wt was significantly reduced in high dose males but not in the recovery group; 

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
there was no effect on food consumption.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
In high dose males water consumption was decreased, however this changes were not seen after the recovery period.

CLINICAL CHEMISTRY
Clinical chemistry showed a significant increase in albumin in both sexes at the highest dose that was not present after recovery.  Males showed a decrease in alpha-1-globulin at the 20 and 100 mg/kg dose levels that was not present in recovery animals. 

URINALYSIS
In high dose males, urine protein was increased, however these changes were not seen after the recovery period.

GROSS PATHOLOGY
In high dose males there was increased brain/body wt ratio and increased kidney/body wt ratio; both effects were absent in recovery males. 

HISTOPATHOLOGY: NON-NEOPLASTIC
Histopathology showed increased hyaline droplets in renal tubule epithelium of all male rats in the 20 and 100 mg/kg groups.  Thyroid effects were hypertrophy of follicular cells, decreased colloid and irregular follicular shape in 6 males, 1 female in 100 mg/kg group, 1 male each in 20 and 4 mg/kg groups.  These thyroid effects were not seen in any recovery animals.

Effect levels

open allclose all
Dose descriptor:
LOAEL
Effect level:
4 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Dose descriptor:
LOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Dose descriptor:
NOAEL
Effect level:
< 4 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Hyaline droplets in the renal tubular epithelium, hypertrophy and irregular shape of follicular cells and decreased colloid in the thyroid, were observed in male rats at doses of 4 mg/kg/day and higher. The kidney effects are likely due to the male-rat-specific alpha 2u globulin accumulation. Increa ed albumin, and hypertrophy of follicular cells and decreased colloid in thyroid were found in the female 100 mg/kg/day group. Thyroid effects are reversible in both sexes. Therefore, the oral NOAEL for systemic effects other than thyroid and kidney is 20 mg/kg/day, based on reduced body we ght of females in the 100 mg/kg/day group.
Executive summary:

A rat 28-day repeated oral dose toxicity test was conducted under the guidelines of the Japanese government (MHLW, Japan, 2000). Sprague-Dawley rats (7 animals/sex/dose with recovery groups of 7 additional animals/sex for the control and high dose groups held an additional 14-d) received doses of 0, 4, 20 and 100 mg/kg/day for 28-d by oral gavage. 

In the 100 mg/kg group females the mean body weight was significantly reduced at dosage termination but not in the recovery group. Food consumption was not affected. Urinalysis revealed an increase in the number of animals with protein-positive urine and a decrease in water consumption in males given 100 mg/kg. These changes were not found at the end of recovery period. In the hematological examination, no effect was observed after administration of ENB. In the blood biochemical examination, 20 and 100 mg/kg group males showed a decrease in alpha-1 globulin level. However, these changes were not found at the end of the recovery period. In high dose males there was increased brain/body wt ratio and increased kidney/body wt ratio; both effects were absent in recovery males. At autopsy, pale discoloration of the kidneys was observed in males given 100 mg/kg, and histopathology showed increased hyaline droplets in renal tubule epithelium of all male rats in the 20 and 100 mg/kg groups. Histopathological examination of the thyroid indicated hypertrophy of follicular epithelium, as well as decrease in colloid or irregular shape of follicles in males given 4 mg/kg or more. Hypertrophy of follicular epithelium and decrease in colloid were also observed in females given 100 mg/kg. There were no effects on the testes. The NOAEL of ENB for repeated dose toxicity was reported to be less than 4 mg/kg/day for males (based on thyroid effects at 4 mg/kg and kidney effects at 20 mg/kg) and 20 mg/kg/day for females (based on thyroid effects at 100 mg/kg). However, the male rat kidney effects are consistent with alpha-2u-globulin nephropathy and are not considered to be relevant to humans (IARC, 1998). The thyroid effects are also likely to be a species specific effect not relevant to humans (IARC, 1998). For these reasons, the oral NOAEL based on systemic effects other than thyroid and kidney is 20 mg/kg/d, based on reduced body weight of females in the 100 mg/kg/d group.