Cycloocta-1,5-diene

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Unnamed

Year:

2002

Reference 2

Reference Type:

publication

Title:

Unnamed

Year:

2001

Materials and methods

Principles of method if other than guideline:

Method: other: See Test Conditions

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Cycloocta-1,5-diene

EC Number:

203-907-1

EC Name:

Cycloocta-1,5-diene

Cas Number:

111-78-4

Molecular formula:

C8H12

IUPAC Name:

(1Z,5Z)-cycloocta-1,5-diene

Details on test material:

1,5-cyclooctadiene supplied by DuPont Nylon (LaPorte, TX, USA), purity >99%

Test animals

Species:

rat

Strain:

other: Crl:CD(R)BR

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ORGANISMS
- Source: Charles River Breeding Laboratories, Raleigh (NC, USA)
- Age: approximately 8 weeks
- Weight at study initiation: 261-312 g
- Number of animals: 10 for standard toxicity + 10 for neurotoxicity = 20  / exposure level

Administration / exposure

Route of administration:

inhalation

Type of inhalation exposure:

whole body

Vehicle:

other: houseline air

Remarks on MMAD:

MMAD / GSD: no data

Details on inhalation exposure:

ADMINISTRATION / EXPOSURE 
- Duration of test/exposure: Five days exposure, two days rest, four days  exposure. Last exposure on day 11, first sacrifice (5 rats per group) on  
day 12, final sacrifice (remaining rats) on day 25.
- Type of exposure: inhalation, whole-body
- Generation of test atmosphere: Addition of heated test substance to  nitrogen stream, mixing with houseline air, feeding into top of exposure  
chamber; monitoring or concentration by GC at approximately 1-hour  intervals during exposure

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The atmospheric concentration of the test substance was determined by gas chromatography at approximately 1 hour intervals during each
exposure.

Duration of treatment / exposure:

6 hours/day

Frequency of treatment:

2 weeks (a total of 9 exposures)

Doses / concentrations

Remarks:

Doses / Concentrations:
0; 50; 150; 500 ppm = 0; 225; 675; 2250 mg/m3
Basis:
nominal conc.

No. of animals per sex per dose:

10 for standard toxicity + 10 for neurotoxicity = 20  / exposure level

Control animals:

yes, concurrent no treatment

Details on study design:

Post-exposure period: 14 days (5 rats/group)

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS AND FREQUENCY: 
- Clinical signs: during exposure, daily throughout and recovery period
- Body weight: daily (before exposure) throughout exposure and recovery  periods
- Haematology: 15 parameters = number of erythrocytes, leukocytes, and  platelets; hemoglobin concentration, hematocrit, mean corpuscular 
volume,  mean corpuscular hemoglobin, and mean corpuscular hemoglobin  concentration; relative numbers of neutrophils, band neutrophils,  
lymphocytes, atypical lymphocytes, monocytes, eosinophils, and basophils. 
- Biochemistry: 17 parameters = activities of alkaline phosphatase,  alanine aminotransferase, aspartate aminotransferase, and sorbitol  
dehydrogenase and serum concentrations of glucose, urea nitrogen,  calcium, phosphate, bilirubin, cholesterol, creatinine, total protein,  
albumin, globulin, sodium, potassium, chloride.
- Urinalysis: day prior to bleeding, 9 parameters = volume, osmolality,  urobilinogen, pH, presence of hemoglobin or occult blood, glucose,  
protein, bilirubin, ketone.

Sacrifice and pathology:

ORGANS EXAMINED AT NECROPSY (MACROSCOPIC AND MICROSCOPIC): 
- Macroscopic: "complete", including 6 rats from each of the  neurotoxicity groups;   weights of liver, kidneys, lungs, testes, and brain
- Microscopic: Samples of 36 tissues = liver, kidneys, lungs, duodenum,  heart, spleen, brain (cerebrum, midbrain, cerebellum, medulla/pons),  
spinal cord, stomach, jejunum, ileum, pancreas, cecum, colon, rectum,  mesenteric lymph node, thymus, adrenal glands, sciatic nerve, thyroid  
gland, parathyroid glands, trachea, esophagus, pharynx/larynx, eyes,  prostate, seminal vesicles, urinary bladder, testes, epididymides,  sternum 
(with bone marrow), nose, and gross lesions were saved. All  tissues from the first sacrifice of 0 and 500 ppm groups were  microscopically 
examined. Nose, pharynx, larynx, lungs, liver, and  kidneys from the first sacrifice of the other groups and from the high  dose recovery group
were also examined microscopically. Additionally,  target organ potential was investigated for the second sacrifice samples  by microscopic 
examination of the kidneys from the 150 ppm group and the  pharynx/lanrynx from the 50 and 150 ppm groups.

Other examinations:

OTHER EXAMINATIONS: Neurotoxicity group
- Functional observational battery (FOB) assessments encompassing  approximately 36 endpoints: After exposures 4 and 9
- Motor activity (MA) evaluations encompassing 2 dependent variables:  After exposures 4 and 9
- Neuropathology evaluation: 6/10 rats after final exposure,  approximately 14 tissue samples saved from the nervous system and  skeletal muscle: 
brain (forebrain, cerebrum, mid-brain, pons, medulla,  and cerebellum), spinal cord (cervical and lumbar), sciatic nerve, tibial  nerve, gasserian 
ganglia, cervical and lumbar dorsal root fibers and  ganglia (DRF&G), cervical and lumbar ventral root fibers (VRF), and  gastrocnemius muscle; 
histopathology for tissues from 0 and 500 ppm  groups. No further evaluations of remaining 4 rats/group.

Statistics:

STATISTICAL METHODS:
- One-way analysis of variance (ANOVA) for body weights (gains + final),  organ weights (abs. + rel. to body / brain), clinical pathology  
measurements
- Dunnett's test for pairwise comparisons
- Least significant Difference (LSD) for pairwise comparison of body  weights & gains
- Cochran-Armitage trend test for increases in the incidence of clinical  observations and for descriptive FOB parameters
- Bartlett's test for homogeneity of variances for clinical pathology and  organ weight data; if significant, followed by Kruskal-Wallis test and  
Mann-Whitney U test; Bartlett's test also for forelimb and hindlimb grip  strength and hindlimb foot splay, followed by ANOVA
- Fisher's exact test with a Bonferroni correction for FOB parameters  which were insignificant in the Cochran-Armitage trend test
- Shapiro-Wilk's Test and Levene's Test for motor activity data
- Significance judged at p<0.05 except for the Bartlett's test with  p<0.005

Results and discussion

Results of examinations

Details on results:

ACTUAL DOSE RECEIVED BY DOSE LEVEL BY SEX:   52 +/- 1.8; 150 +/- 0; 500 +/- 9.2 ppm (mean analytical)
TOXIC RESPONSE/EFFECTS BY DOSE LEVEL: 
- Mortality and time to death: No mortalities reported
- Clinical signs: Absence of alerting response toward the end of the  daily exposures in 500 ppm group, recovery within 1/2 hour after  exposure. 
No other test substance-related changes were observed.
- Body weight gain: No test substance-related changes were observed.
- Clinical chemistry: No toxicologically important changes
- Haematology: No toxicologically important changes
- Urinalysis: pH decreases in 500 ppm group at the end of the exposure  period (5.9 vs 6.7 in controls), not after the recovery period; no other  
toxicologically important changes.
- Organ weights: Increased kidney weights in 500 ppm group (+17% relative  to body weight, +15% rel. to brain weight, both statistically  significant;  +13% absolute, not statistically significant) immediately  after exposure, along with hyaline droplets in the kidneys (5/5,  minimal  to moderate), 
reversible after the recovery period
- Gross pathology: No test substance-related changes were observed.
- Histopathology: Minimal to mild degeneration / necrosis of nasal  olfactory epithelium after the exposure period in the 500 ppm group  (4/5); 
minimal to mild degeneration / regeneration in this area after the  recovery period (4/5)
- Other: * There was an increase in the number of rats found sleeping in 150 and  500 ppm groups (6/10 and 6/10, respectively, vs. 2/10 in control)  after  the last exposure, not considered to be an adverse effect. * In the 500 ppm group, the incidences of curled-up posture (6/10 vs 2/10  in 
control) and vocalizations when handled (4/10 vs 0/10) were increased  significantly. * There were no treatment-related effects in the motor activity   evaluation. * In the neuropathologic evaluation, a single "mild" lesion in the lumbar  dorsal root fiber in one 500 ppm group rat was considered not 
to be  compound-related.

Effect levels

1

Dose descriptor:

NOEC

Effect level:

675 mg/m³ air

Sex:

male

Basis for effect level:

other: see: Details on results

2

Dose descriptor:

LOEC

Effect level:

2 250 mg/m³ air

Sex:

male

Basis for effect level:

other: see: Details on result

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

no further remarks

Applicant's summary and conclusion

Conclusions:

Based on the decreased altering response observed in rats during exposure at 500 ppm, and on the effects observed in the nose, kidney and urine in rats at this concentration, the no-observed-effect-level (NOEL) in this study was considered to be 150 ppm.

Executive summary:

In this two week inhalation study, male rats were exposed to 0, 50, 150 and 500 ppm 1,5 -cyclooctadiene. 10 rats per group were used

for standard toxicological evaluations and 10 rats/group were used for neurotoxicity testing. No mortalities were reported. In rats

exposed to 500 ppm there was an absence of alerting response toward the end of the daily 6-hours exposures. These rats appeared to

recover within 1/2 hour after exposure. No other test substance-related changes in clinical signs or body weight parameters were 

observed. Clinical chemistry and haematology showed no toxicologically important changes. The urinalysis demonstrated an pH decreases in 500 ppm group at the end of the exposure period, but  not after the recovery period. Histological effects

attributable to the test item were found in the nose and kidneys of the high dose group. There was a degeneration/necrosis of nasal olfactory epithelium observed immediately after the exposure period and a degeneration/regeneration in this area observed after the 2 -week recovery. In addition , there were increased kidney weights in the 500 ppm group immediatley after exposure, along with increased hyaline droplets in the kidneys, reversible after the recovery peroid.

Based on the decreased altering response observed in rats during exposure at 500 ppm, and on the effects observed in the nose, kidney and urine in rats at this concentration, the no-observed-effect-level (NOEL) in this study was considered to be 150 ppm.