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Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study to GLP but original study report not available.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1997

Materials and methods

Principles of method if other than guideline:
This study was conducted to meet the criteria of the following guidelines: Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA), Toxic Substances Control Act (TSCA), Food and Drug Administration (FDA), Organisation for Economic Co-operation and Development (OECD), Ministry of Agriculture, Forestry and Fisheries (MAFF), and European Economic Community (EEC).
GLP compliance:
yes
Remarks:
Test laboratory not specified in publication but thought to be Union Carbide Corporation, USA
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)
- Purity: 99.3%, No compositional changes were measured over the period of the study.
- Source: Union Carbide Corporation, South Charleston, West Virginia

Test animals

Species:
rat
Strain:
other: CDF
Sex:
male/female
Details on test animals and environmental conditions:
Each rat was uniquely numbered by toe-clipping.

TEST ANIMALS
- Source: Charles River Laboratories (Kingston, NY).
- Age at study initiation: 28 days
- Housing: individually in wire-mesh cages
- Diet: powdered, certified AGWAY~ PROLAB~ Animal Diet (Agway Inc.) ad libitum during non-exposure periods.
- Water: tapwater.
- Acclimation period: The animals were acclimated to the exposure chamber for two days prior to start of the exposures.

ENVIRONMENTAL CONDITIONS
- The room temperature and relative humidity were monitored continuously.
- Temperature: 20.0-24.5°C.
- Relative humidity: 40-64%
- Photoperiod: 12-hour.

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: Not applicable
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: stainless steel chambers with glass windows; The volume of each chamber was approximately 900 liters
- System of generating particulates/aerosols: Liquid ENB was metered from syringe pumps. The temperature in each evaporator was maintained at a level sufficient to vaporize the test substance (33-77 Celsius).
-
- Air flow rate: 200 liters/minute
- Air change rate: (approximately 13 air changes per hour).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chamber concentrations of ENB vapor were analyzed by gas chromatography. At least two samples per hour were obtained from each exposure chamber and the control chamber. Analysis was by GC/FID
Duration of treatment / exposure:
At least 13 weeks. During the 14th week, the males and females (including those animals designated for perfusion-fixation) received two and three consecutive days of exposure, respectively. The animals assigned to the recovery period were exposed three consecutive days during the 14th week.
Frequency of treatment:
6 hours per day, 5 days per week. Total exposures males, 66; females 67.

Doses / concentrations
Remarks:
Doses / Concentrations:
0 (control), 5, 25, and 150 ppm (target), 5, 25, and 149 ppm (mean analytical); (24, 122, 732 mg/m3)
Basis:
analytical conc.
No. of animals per sex per dose:
15 male and 15 female

Control animals:
other: yes, filtered air
Details on study design:
Animals were assigned to three exposure groups and an air-exposed control group. Fifteen male and 15 female rats were assigned to each group. 10 animals from each sex group were sacrificed during week 14 and the remaining 5 males, 5 females per group followed for 4-wk recovery period with no exposure before sacrifice.
Positive control:
No

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were individually observed for signs of toxic effects except during the exposures. During the exposures, observations were recorded on a group basis. Preceding and following each exposure, observations were recorded for animals exhibiting clinical signs. On nonexposure days and during the recovery period, animals were observed once a day for clinical signs.

BODY WEIGHT: Yes
- All animals were weighedprior to the first exposure. The animals were weighed weekly.The animals assigned to the 4-week recovery period were weighed once a week and immediately prior to sacrifice.

FOOD AND WATER CONSUMPTION:
- Food and water consumptions were measured weekly for all rats during the exposure regimen and the 4-week recovery period.

HAEMATOLOGY: Yes
- Parameters checked: total leukocyte count, erythrocyte count, hematocrit, hemoglobin, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin, concentration (MCHC), platelet count, prothrombin time, activated partial, thromboplastin time

CLINICAL CHEMISTRY: Yes
- Parameters checked: glucose, urea nitrogen, creatinine, total protein, albumin, globulin, total, conjugated, and unconjugated, bilirubin, calcium, phosphorus, sodium, potassium, chloride, total cholesterol, Free T3, Total T3, T3 uptake, Free T4, Total T4, aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatine kinase (CK), lactate dehydrogenase (LOH), gamma-glutamyl transferase (GGT), sorbitol dehydrogenase (SOH), alkaline phosphatase (ALK), glutamate dehydrogenase (GLOH). Thyroid hormones were measured by commercially available RIA.

URINALYSIS: Yes
- Parameters checked: urine volume, appearance, sediment (microscopic), protein, potassium, osmolality, pH, occult blood, glucose, creatinine, chloride, protein, bilirubin, urobilinogen, ketones, sodium

Sacrifice and pathology:
The male and female animals were sacrificed after 66 and 67 exposures respectively. Rats were killed by exsanguination via the brachial blood vessels following anesthesia with methoxyflurane.

GROSS PATHOLOGY: Yes, The brain, liver, kidneys, lungs, spleen, heart, thymus, pituitary, thyroid (weighed after fixation), adrenal glands from all animals, and testes from all males were weighed at sacrifice.
HISTOPATHOLOGY: Yes, Selected tissues were fixed in 10% neutral buffered formalin. (except for eyes and testes which were fixed in Bouin's solution).
Other examinations:

- Morphometric analysis was conducted on thyroids from 10 males in the control and all exposure groups. Morphometric studies were performed to measure changes in the quantity of follicular colloid of the thyroid glands.
Statistics:
Quantitative continuous variables were intercompared between ENB groups and the corresponding air-control groups using Bartlett's homogeneity of variance, analysis of variance, and Duncan's multiple range tests. Non-parametric data were statistically analyzed using the Kruskall-Wallis test followed by the Mann-Whitney U test. A fiducial limit of <0.05(two-tailed)was used as the critical level of significance for all comparisons.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
ACHIEVED EXPOSURES
Means of levels determined at 5 sites in the chamber were 4.27 +/- 0.28, 24.5 +/- 098 and 150 +/- 5.1 ppm with coefficients of variation of 6.6, 4.0 and 3.4% around the target concentrations (5, 25 and 150 ppm) indicating a uniform ENB vapour distribution.

CLINICAL SIGNS AND MORTALITY
No animals died during the study. Periocular swelling and/or encrustation was seen in females of all groups and males of the 149 ppm group.  Corneal dystrophy (linear opacity) was seen with equal frequency in both ENB and air control groups. 

BODY WEIGHT AND WEIGHT GAIN
There were no effects on absolute body weight gain. Body weight gains for the 24.8 and 149 ppm groups of male rats were 13% and 21% lower, respectively, than the controls at the end of the first exposure week, with subsequent recovery.

FOOD CONSUMPTION
During the 14-wk exposure, food consumption was increased in the 149 ppm group for most periods for males and females and remained increased during the recovery period compared to controls.

WATER CONSUMPTION
Water consumption was increased in both males and females in the 149 ppm group during exposure and recovery periods. Increases ranged from 4.9 to 23.3% for males and -1.8 to 17.6% for females during exposure and from 14.9 to 20% for males and from 2.6 to 13.1% for females during the recovery period.

HAEMATOLOGY
Top dose males had statistically significantly decreased erythrocyte count, hemoglobin concentration, and hematocrit; increased MCHC was seen in the middle dose .  Males in the low dose group had decreased erythrocyte count and hematocrit, with increased MCHC.  No statistically significant effects were seen in the recovery group.

CLINICAL CHEMISTRY
Other than small changes in certain thyroid hormone concentrations there were no statistically significant effects on serum chemistry.  Serum T3 uptake was slightly reduced, with statistical significance, in 24.8 and 149 ppm group males at the 14-wk sacrifice, but not at the recovery sacrifice. Serum T4 was significantly raised in middle-dose males in the 9 day study at not significantly at the top dose.

URINALYSIS
The only findings were statistically significant decreased urine osmolality (15%) and creatinine (20.8%) at the 14-wk sacrifice, but not the recovery sacrifice, in the male rats exposed to 149 ppm ENB.

GROSS PATHOLOGY
Relative liver weights were increased only in the 149 ppm males, and no histopathological findings were observed. Relative kidney weights were increased for males of all ENB-treatment groups and females exposed to 149 ppm ENB.  Absolute and relative thyroid gland weights were increased for males of the middle- and top-dose groups in the 9-day study. There were no effects on thyroid gland weight in the subchronic study.

HISTOPATHOLOGY: NON-NEOPLASTIC
Other than the thyroid gland, no exposure related lesions were observed.  Principal target organ effects were to the thyroid gland, which showed an exposure concentration-related, but not exposure time-related, depletion of follicular colloid that resolved during the recovery period.  There was light microscopic evidence for a hypertrophic and hyperplastic response in the follicular epithelium that resolved more slowly.  The thyroid colloid depletion was a graded effect without a clear no-effect concentration, but was not accompanied by any clinical or clear biochemical evidence for thyroid dysfunction.  The depletion was generally not seen in the recovery animals.  A no-effect concentration of 4.9 ppm was established for the follicular cytological effects.

Effect levels

open allclose all
Dose descriptor:
NOAEC
Effect level:
5 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: reversible thyroid follicular cytology
Dose descriptor:
NOAEC
Effect level:
25 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: other than thyroid
Dose descriptor:
LOAEC
Effect level:
150 ppm
Based on:
test mat.
Sex:
male/female

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Morphometry: A statistically significant reduction in mean colloid area per follicle was measured in rats of the high dose group (150 ppm) as compared to control group animals. There were no significant changes in the mean colloid area of recovery group animals of the high concentration group.

Applicant's summary and conclusion

Conclusions:
Exposure of rats to ENB at 5, 25 and 150 ppm for 13+ weeks produced no mortality and minor systemic toxicity. No histopathologic findings, other than in the thyroid gland, were found. A no-effect concentration of 4.9 ppm ENB was established for the follicular cytological effects. The NOAEL for effects other than thyroid is 25 ppm (122 mg/m3).
Executive summary:

Ballantyne et al. (1997) published a 14-week inhalation study of ENB in rats conducted by guidelines and GLP. CDF rats (15/sex/dose) were exposed to analytically measured ENB vapor concentrations of 0, 5, 25, or 149 ppm (0, 24, 122, 732 mg/m3, respectively) for 6 hr/day, 5 days/week for 13+ weeks (a total of 67 exposures). No deaths occurred during the study. Overall, toxicity was greater in males than in females. Clinical chemistries were unaffected with the exception of reduced tri-iodothyronine (T3) uptake in males of the 25 and 149 ppm groups at 14 wk by not after the 4-wk recovery. T3 uptake was slightly reduced (not statistically significant) in male rats of the 5 ppm exposed group only at 14 wk. Urine osmolality and creatinine were decreased in males of the 149 ppm group at wk 14, but not in the recovery animals. Urine protein was increased in the 149 ppm exposed females at 14 wk and after the 4-wk recovery. Relative kidney weights increased in all groups of males and the females exposed to 149 ppm ENB. Relative kidney weights were increased (not statistically significant) in 149 ppm exposed males at the end of the 4-wk recovery period. Relative liver weight was increased in 149 ppm exposed males, and also at the end of the recovery period. Relative liver weight was increased in 149 ppm exposed females only after the recovery period. Principal target organ effects were to the thyroid gland, which showed an exposure concentration-related, but not exposure time-related, depletion of follicular colloid that resolved during the recovery period in all exposed groups except the 5 ppm exposed females. There was light microscopic evidence for a hypertrophic and hyperplastic response in the follicular epithelium that resolved more slowly. The thyroid colloid depletion was a graded effect without a clear no-effect concentration, but was not accompanied by any clinical or clear biochemical evidence for thyroid dysfunction. The depletion was generally not seen in the recovery animals. A no-effect concentration of 5 ppm (24 mg/m3) was established for the follicular cytological effects. Other than the thyroid gland, no exposure related lesions were observed following histopathology, therefore, for effects other than thyroid, the NOAEL is 25 ppm (122 mg/m3) based on changes in organ weights and urinalysis findings observed at 149 ppm (732 mg/m3).