Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1996-05-28 to 1996-07-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
cis,cis-1,5-cyclooctadiene of Hüls AG. Purity > 99 %. ID 0637/81751, produced Nov 1994.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ORGANISMS
- Strain: Wistar (Hsd/Win:WU)
- Source: Harlan Winkelmann GmbH, 33176 Borchen (Germany)
- Age: 6-8 weeks
- Weight at study initiation: females mean 118 g, males mean 141 g
- Number of animals: 5 per dose group and sex, total 60 including  satellite groups
- Diet: ad libitum, R10 special diet for rats, SSniff R
- Water: ad libitum, tap water
- Acclimation period: at least 5 days
ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 3 °C
- Humidity: 30 - 70 %
- Photoperiod: 12 hours artificial light, 12 hours dark
- Air changes: 15 per hour

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
ADMINISTRATION / EXPOSURE 
- Vehicle: corn oil
- Concentration in vehicle: 0 / 25 / 75 / 225 g/l
- Total volume applied: 2 ml/(kg bw * day)
Details on analytical verification of doses or concentrations:
ACTUAL DOSE RECEIVED BY DOSE LEVEL BY SEX:   Good stability of the test substance and a satisfactory concordance  between measured and nominal concentrations in the vehicle solutions were  found.
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
daily including weekends
Doses / concentrations
Remarks:
Doses / Concentrations:
50, 150, and 450 mg/(kg bw * d)
Basis:
nominal in diet
No. of animals per sex per dose:
5 , total 60 including  satellite groups
Control animals:
yes, concurrent vehicle
Details on study design:
Post-exposure period: 14 days (satellite groups) / 2 days (other groups)
Positive control:
no positive control

Examinations

Observations and examinations performed and frequency:
SATELLITE GROUPS AND REASONS THEY WERE ADDED: Control and high dose  satellite groups for recovery studies
CLINICAL OBSERVATIONS AND FREQUENCY: 
- Clinical signs: recorded twice (weekends: once) daily (approximately 1  or 2 hours after application considering the peak period of the clinical  
symptoms); detailed clinical examination once a week.
- Mortality: twice (weekends: once) daily
- Body weight: at weekly intervals from prior to first dose until days of  necropsy.
- Food consumption: cagewise (= groupwise, i.e. for 5 animals of same sex  and dose level) at weekly intervals
- Water consumption: observed cagewise, daily
- Ophthalmoscopic examination: during acclimatization and before terminal  bleeding in the high-dose and control animals. An ophthalmoscope was  used  and one eye was treated with Mydriaticum Stulln(R) prior to investigation.
- Hematology: end of treatment or recovery period: Red blood cell count  (RBC); total white blood cell count (WBC); platelet count (PLT);  hemoglobin 
(HGB); hematocrit (HCT); erythrocyte mean corpuscular volume  (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin  
concentration (MCHC); differential white blood cellcount (neutrophils  NEUTRO, basophils BASO, lymphocytes LYMP, monocytes MONO, plasma 
cells  Plas, unclassifiable cells UNCLC).
- Biochemistry: end of treatment or recovery period: Sodium; potassium;  calcium; aspartate aminotransferase (AST); alanine aminotransferase  (ALT);  alkaline phosphatase (AP); glucose (GLUC); triglycerides (TRIG);  cholesterol (CHOL); total bilirubin (TBIL); blood urea nitrogen (BUN);  creatinine 
(CREA); total protein (TPROT); albumin (ALB).
- Urinalysis: end of treatment or recovery period: Volume (VOL); specific  gravity (SPGR); pH; color.   
Semiquantitative: Protein (PROT); glucose (GLUC); keton; urobilinogen  (UBG); blood ingredients.    
Microscopical urine sediment analysis: Leucocytes (LEUCO); erythrocytes  (ERY); bacteria (BAC); epithelial cells, squamous (ECSQ); oxalate  
crystals (OXA); triple phosphate crystals (TRIP); urate crystals (URA);  carbonate (CARB); granular cylinders (GACYL)
Sacrifice and pathology:
ORGANS EXAMINED AT NECROPSY (MACROSCOPIC AND MICROSCOPIC): 
- Macroscopic: Complete autopsy incuding macropathological examination on  all animals including satellite groups.   Weights of adrenals, kidneys, 
liver, spleen, testes.
- Microscopic: preparation of samples of adrenals, aorta (thoracic),  anus, brain, cecum, coagulation gland, colon, concha (tattooed),  duodenum, 
epididymides, eyes, exorbital lacrimal glands, gross lesions,  heart, ileum, sternum, jejunum, kidneys, larynx, liver, lungs, lymph  nodes (skin, 
cervical & mesenteric), mammary gland, muscle (skeletal),  ovaries, esophagus, pancreas, pituitary, prostate, rectum, salivary  glands, sciatic nerve,  seminal vesicles, skin, spinal cord (cervical),  spleen, stomach, testes, thymus, thyroid / parathyroid, tongue, trachea,  urinary bladder, 
uterus / vagina;   examination for selected high-dose and control animals + examination of livers (= target organ) of all other animals and lesions  
related to application failure.
Other examinations:
no other examination
Statistics:
STATISTICAL METHODS: (disregarding animals that died during the study)
- Kruskal Wallis non parametric analysis of variance and in case of  significance Wilcoxon, Mann, and Whitney U-test: absolute body weights  and 
their changes; absolute and relative organ weights; differential  blood count; urinalysis data.
- One way analysis of variance (ANOVA) incorporating a Bartlett's test  for homogeneity of variance and in case of heterogeneous variances a  
Kruskal Wallis test / in case of significant ANOVA a Scheffe test:  Hematological data (except differential blood count) and serum clinical  chemistry  data.
- Group means and standard deviations or medians where appropriate.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Details on results:
TOXIC RESPONSE/EFFECTS BY DOSE LEVEL: 
- Mortality and time to death:   Four animals (all differing in dose level and/or sex) died during the  treatmend period. These mortalities were not 
attributed to the test  substance but to application failure (3 animals), or could not be  investigated due to total cannibalism (1 control female).
- Clinical signs:   At the beginning of the treatment period clinical signs were observed  in all dose groups after application of the test substance. 
Part of the  symptoms was probably caused by application failure, which was found upon  necropsy to have occurred frequently in this study. The 
most severe  clinical signs were seen in the high dose groups. The predominant  clinical symptom was lethargy sometimes associated with ataxia, 
abnormal  gait, closed eyes or piloerection. These symptoms disappeared a few hours  after treatment and the animals showed nothing abnormal. 
At the end of  the treatment period animals of all dose groups showed less or no  clinical symptoms after application of the test substance. As the  
symptoms described were not observed in the control groups, they were  considered to be related to the administration of test substance.   During 
the recovery period, none of the above clinical symptoms were  observed (apart from piloerection in the first two days).
- Body weight gain:   Affected in high-dose females (total gain -17.0 %, not statistically  significant) and males (-30.6 %, p < 0.05).   These changes
were considered to be test substance related. Evidence of  reversibility was found during recovery period.
- Food/water consumption:   
The high dose male group (+36.8 %) and the high and medium dose female  group (+14.3 %) showed an increased food conversion rate 
(not identified  as statistically significant). These findings were considered to be test  substance related. Evidence of reversibility was found.   No 
overt intergroup differences in water consumption were noted.
- Ophthalmoscopic examination:   No signs of test substance related effects on lenses or cornea were  observed in the high dose group.
- Clinical chemistry:   All statistically significant differences between treated and control  were low, within the normal range of historical background 
data, and  inconsistent between males and females. Therefore they were not  considered to be of toxicological importance.
- Hematology:   As with clinical chemistry, no findings of toxicological importance  were noted in treated animals.
- Urinalysis:   The only statistically significant observation was a minor increase in  pH in high-dose recovery males, which remained within the range  of  historical controls.
- Organ weights:   Liver (relative): Significantly (p < 0.05) increased in males (mid-dose  +13.3 %; high-dose +22.4 %) and females (mid-dose +18.8 %,  high-dose  +31.1 %).   Kidney (relative): Significantly (p < 0.05) increased in high-dose  females (+12.6 %).   These effects were considered to be 
test substance related. Evidence of  reversibility was found at the end of the recovery period.   Other organs: No significant findings.
- Gross pathology:   No macroscopical lesions were considered to be related to the test  item. Necropsy revealed mild to severe lesions in the 
thoracal cavities  of five animals, which were assigned to application failure.
- Histopathology:   In line with the increase in liver weight, hepatocellular hypertrophy  without any degenerative signs was noted in mid- and 
high-dose male and  female rats and in low-dose males. Evidence of reversibility for these  histopathological findings was found at the end of the 
recovery period.  There were no significant histopathological findings in correlation with  the increased relative kidney weight in high-dose females.   Other histopathological findings consisted of mild to severe lesions  caused by application failure and spontaneous lesions in the male and  female 
animals of all groups such as hydrometriosis of the uterus,  calcification of the kidney, spermatic granuloma of the epdidymis and  multifocal 
lymphocytes in the lung respectively pneumonitis.

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: see: Details on results
Key result
Dose descriptor:
LOAEL
Effect level:
150 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: see: Details on results

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

no further results


Applicant's summary and conclusion

Conclusions:
For all the effects observed, evidence of reversibility was found after two weeks without treatment.
It is suggested that the reversible liver changes observed are an expression of an adaptive response of the liver to the test substance. As such, it is
generally not considered as an adverse effect (Popp JA and Cattley RC (1991). Hepatobiliary system. In: Haschek WM and Rousseaux CG (Eds),
Handbook of toxicologic pathology, p. 279-315. Academic Press Inc., San Diego).
Therefore, in the experimental conditions of the study, the no-observed-adverse-effect level (NOAEL) is 50 mg/kg/day.
Executive summary:

The daily administration of the test item 1,5 -Cyclooctadien by oral route to Wistar rats at nominal concentrations of 50, 150 and 450 mg/kg/day induced dose related clinical symptoms. The observation of the animals was protocolled approximatley 1 or 2 hours after application considering the peak period of the clinical symtoms. These symptoms disappeared a few hours later and the animals showed nothing abnormal. The predominant clinical symptom was lethargy sometimes associated with ataxia, abnormal gait, closed eyes or  piloerection. At the end of the treatment period animals of all dose groups showed less or no clincal symptoms after application of the test substance. During treatment period the high dose male and female group showed a reduction in bodyweight and an increased food conversion rate and an increase in relative liver weights. High dose females showed also an increased relative kidney weight. In line with the increase in relative liver weight hepatocellular hypertrophy was noted in high dose male and females. The male and female medium dose group showed also hepatocellular hypertrophy. All these findings were considered to be test substance-related.

For all the effects described evidence of reversibility was found after two weeks without treatment. It is suggested that the reversible liver changes observed are an expression of an adaptive response of the liver to the test substance. As such, it is generally not considered as adverse effect (Popp JA and Cattley RC (1991).

Therefore, in the experimental conditions of the study, the no-observed-adverse-effect level (NOAEL) is 50 mg/kg/day.