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Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP, Near-guideline, animal experimental study, also published in peer-reviewed literature, minor restrictions in design and reporting but otherwise acceptable for assessment.
Cross-reference
Reason / purpose:
reference to same study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1982
Reference Type:
publication
Title:
Subchronic Toxicity Study Of Dicyclopentadiene Vapour In Rats
Author:
Bevan C, Snellings W, Dodd D and Egan G
Year:
1992
Bibliographic source:
Toxicol. Ind. Health Vol 8 (6) 353-367

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): dicyclopentadiene (DCPD)
- Source: Exxon Chemical Company, Baton Rouge, LA, USA
- Sample reference: BRRC 43-156
- Physical state: clear, colourless liquid
- Analytical purity: ≥95% endo-DCPD, 0.5% exo-DCPD
- Impurities (identity and concentrations): several impurities of which only cyclopentadiene. and isoprene were present at ≥0.5%
- Stability under test conditions: The composition remained stable throughout the study

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratory (Portage, MI, USA)
- Age: 30-34 days old on receipt
- Health assessment: confirmed following arrival
- Housing: 3/sex/cage during non-exposure period, individually during exposure, in suspended, stainless-steel cages
- Diet: NIH-07 diet ad libitum except during exposure
- Water: ad libitum except during exposure
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS (ANIMAL ROOM)
- Temperature: 20-22°C
- Humidity: 40-60%
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: June 25, 1980 To: January 16, 1981

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 4.3 m3 stainless-steel and glass inhalation chambers
- System of generating atmosphere: Liquid dicyclopentadiene was metered from either a piston or syringe pump assembly into a heated, spiral-grooved Pyrex tube and mixed with air entering the bottom of the tube at a flow rate of approximately 2000 L/min.
- Complete vaporization of dicyclopentadiene was achieved while the temperature was kept below 35°C the point at which heat fracturing occurs producing the monomer.

TEST ATMOSPHERE
- Brief description of analytical method used: Air samples assayed using a Perkin Elmer 3920B dual column gas chromatograph equipped with a hydrogen flame ionization detector and a linear temperature programmer.
- Samples taken from breathing zone: yes
- The column was a 5 ft x 1/4 inch O.D. stainless-steel column packed with 20% SP2100 on Supelcoport (80-100 mesh) operating at 150°C.
- The nitrogen carrier flow rate was 75 mL/min, the hydrogen flow rate was 60 mL/min, and the air flow was 475 mL/min.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The chamber concentration of dicyclopentadiene was measured six times per day for each exposure group.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hours/day, 5 days/week for up to 13 weeks
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 1, 5, or 50 ppm
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0.0, 1.0, 5.1 and 51 ppm
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
0, 5, 27.6, 276 mg/m3
Basis:
analytical conc.
No. of animals per sex per dose:
51
Control animals:
yes, concurrent vehicle
Details on study design:
- Post-exposure recovery period in satellite groups: up to 13 weeks
- Animals killed following completion of exposure at 2, 6, or 13 weeks and at postexposure weeks 4 or 13

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: before and after each exposure and daily (5 days/week) during the recovery period

BODY WEIGHT: Yes
- Time schedule for examinations: prior to the first exposure; weekly during the first 4 weeks of exposure and every 2 weeks thereafter; the first 5 weeks of the recovery period, and then every two weeks. All animals weighed prior to termination.

FOOD CONSUMPTION: Yes
- Frequency: during each urine collection period

WATER CONSUMPTION: Yes
- Frequency: during each urine collection period

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to sacrifice
- Dose groups that were examined: High dose only in the first instance, intermediate dose and control group depending on findings

HAEMATOLOGY: Yes
- Time schedule and numbers of animals for collection of blood: all animals prior to being killed after 2, 6 and 13 weeks of exposure, and after 4 and 13 weeks post-exposure.
- Anaesthetic used for blood collection: Yes (methoxyflurane)
- Animals fasted: No
- Parameters examined: Erythrocyte count, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration and total /differential white blood cell counts.

CLINICAL CHEMISTRY: Yes
- Time schedule and numbers of animals for collection of blood: all animals prior to being killed after 2, 6 and 13 weeks of exposure, and after 4 and 13 weeks post-exposure.
- Anaesthetic used for blood collection: Yes (methoxyflurane)
- Animals fasted: No
- Parameters examined: creatinine, urea nitrogen, calcium, phosphorus, chloride, alanine aminotransferase, aspartate aminotransferase, total protein, albumin, total bilirubin, alkaline phosphatase, glucose and osmolality.

URINALYSIS: Yes
- Time schedule for collection of urine: weekly for the first 4 weeks of the study and prior to euthanasia.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters examined: pH, protein, glucose, bilirubin, urobilinogen, blood, urine volume, specific gravity, osmolality, colour and turbidity, creatinine, urea nitrogen, calcium, phosphorus, chloride, sodium, potassium and microscopic analysis.
- A urinary concentration test was performed on those rats selected for sacrifice at the end of the 13-week recovery period. The test was done on Day 6 (males and females) and on Day 83 (males only) of the recovery period, and involved the collection of urine samples from rats that had been deprived of water for 16 hours. Urine samples were then collected over a 6-hour period during which the animals were deprived of both food and water.

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. All animals
ORGAN WEIGHTS: Yes. Kidneys, lung, liver and testes
HISTOPATHOLOGY: Yes. The following tissues were taken and fixed: Kidneys, liver, testes, adrenals, bone and bone marrow (sternal), brain (brain stem, cerebellum, cerebrum), epididymides, eyes, heart, kidneys, larynx, liver, lungs, lymph nodes (mediastinal), muscle (gastrocnemius), nasal turbinates, parathyroids, pituitary, sciatic nerve, spleen, testes, thymus, thyroids, trachea, urinary bladder, and gross lesions. All tissues from the high-exposure and control groups were stained with haematoxylin and eosin (H&E) and examined. In the mid and low groups only kidneys and urinary bladders were examined. Kidneys and urinary bladders were stained with periodic acid and H&E.
ELECTRON MICROSCOPY: Three rats/sex/exposure group were killed at week 13 and at the end of the recovery period, and the kidneys were removed for electron microscopic evaluation.

Statistics:
Bartlett's test of homogeneity of variance to determine if the groups had equivalent variances. If the variances were not significantly different, the groups were compared using analysis of variance (ANOVA). If significant differences among the means were indicated, the Duncan's multiple range test was used to determine which dicyclopentadiene-treated groups differ from the controls.

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
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
WATER CONSUMPTION: In male rats, mean water consumption was significantly increased at Weeks I and 13 at 1 ppm; Week 13 at 5 ppm; and on multiple occasions, including post-exposure at 50 ppm. In female rats, mean water consumption was significantly increased at 5 ppm and 50 ppm at Weeks 13 and 50 ppm at Week 19.

URINALYSIS: Epithelial cells were seen in urine of exposed male rats: the number of epithelial cells and the number of affected animals increased during the exposure period, but were not present at 13 weeks post-exposure. Epithelial cell casts also seen in urine sediment of treated male rats during exposure but not during the recovery period. After 1 week exposure, males at 50 ppm showed decreased specific gravity and osmolality, and increased volume. These effects increased in severity during the exposure period. At the end of Week 13, urine osmolality had decreased by 14% and 32% compared to controls at 5 and 50 ppm respectively. During the recovery period, the alteration in urine osmolality and specific gravity became less apparent but still persisted in the high-dose group even after 92 days post-exposure. At 92 days post-exposure, urine osmolality at 50 ppm was 14% decreased compared to controls.

When rats were deprived of water overnight prior to urine collection, the osmolality of male rats exposed to 5 and 50 ppm of DCPD was significantly decreased (94% and 69% respectively of unexposed male rats). This effect was specific only to male rats. After 83 days postexposure, the impaired urine concentrating ability of the kidney had improved, a difference in urine osmolality was evident only in male rats exposed to 50 ppm (87% of control).

The urinary excretion rate of Na+ in male rats exposed to 5 or 50 ppm DCPD was significantly reduced as compared to control animals, whereas the urinary excretion rate of K+ was significantly elevated at 50 ppm. These changes were first observed after two weeks of exposure and persisted throughout the exposure period. Urinary excretion rates returned to control values after a recovery period of 4 weeks.

ORGAN WEIGHTS: Relative mean liver weights in male rats exposed to 50 ppm were significantly increased compared to controls. In male rats exposed to 5 ppm DCPD for 13 weeks, the absolute mean and the relative mean kidney weights were decreased when compared to controls. These differences in organ weights disappeared during the recovery period.

HISTOPATHOLOGY: NON-NEOPLASTIC: Male rats exposed to 5 and 50 ppm DCPD accumulated hyaline droplets in the proximal convoluted tubular epithelial cells to a much greater extent than in control rats. This accumulation of hyaline droplets occurred as early as the end of two weeks of exposure and throughout the exposure period, but were not observed during the postexposure or recovery period. Males exposed to 1 ppm DCPD sacrificed after Week 6 had a higher incidence of hyaline droplets than at Week 13. Intraluminal protein was also observed in DCPD-treated male rats as early as Week 2. By Week 13, all male rats exposed to 50 ppm had tubular proteinosis. However, unlike the hyaline droplets, there was incomplete recovery during the postexposure period. Similar results were observed for the treatment-related increase in regenerative epithelium which increased in severity over the exposure period, lessening only slightly during the recovery period. During the postexposure period, the incidence of regenerative
epithelium also increased in both exposed and nonexposed female rats. Other histologic changes observed in control and treated male rats included
glomerular basement membrane thickening and interstitial nephritis, which increased in incidence during both the exposure and recovery period. Histological examination of other organs and tissues in rats did not reveal any treatment-related changes.

ELECTRON MICROSCOPY: Electron dense crystalline material within hyaline droplets from proximal tubular cells of DCPD-exposed male rats was seen. These structures were absent in proximal tubular cells of control males. After the 13-week recovery period, these electron dense structures were not observed in the proximal cells of rats from the high-dose group.

Effect levels

Dose descriptor:
NOAEC
Effect level:
50 ppm
Sex:
male/female
Basis for effect level:
other: 276 mg/m3. No systemic toxicity at highest dose tested

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Dicyclopentadiene produced kidney damage in male rats at all dose levels. There were epithelial cells excreted in the urine and alterations in kidney structure in the proximal tubule, such as an increase in the incidence of hyaline droplets, regenerative epithelium, and an accumulation of tubular proteinaceous material. From electron micrographs, many of the hyaline droplets in the exposed male rats appeared electron-dense and angular or crystalline-shaped. These kidney effects were not observed in any of the female rats and were not observed post exposure or at the end of the recovery period.

Incidence and Severity of Hyaline Droplets in Proximal Tubules of Male Rats Exposed to DCPD (Bevan et al 1992)

Week 6

Week 13

Severity*

Control

1 ppm

5 ppm

50 ppm

Control

1 ppm

5 ppm

50 ppm

Mild

0/9

5/9

4/9

0/9

0/9

0/9

8/9

0/9

Moderate

0/9

2/9

1/9

6/9

0/9

0/9

0/9

3/9

Marked

0/9

0/9

0/9

1/9

0/9

0/9

0/9

6/9

* values represent the incidence of structural change at the respective degree of severity.

Applicant's summary and conclusion

Conclusions:
Subchronic exposure of rats to dicyclopentadiene for 13 weeks resulted in no systemic toxicity at 50 ppm. The only chnage observed was a male, rat specific nephropathy, that is characteristic of the hyaline droplet nephropathy produced by a diverse group of compounds. The NOAEC for males and females was reviewed by Bevan et al, 1992 and was concluded to be 5.1 ppm (27.6 mg/m3) for males (excluding the Hyaline droplet effect) and 51 ppm (276 mg/m3) for females.
Executive summary:

Fischer 344 rats were exposed by inhalation to 0, 1, 5 or 50 ppm dicyclopentadiene vapour 6 hr/day, 5 days/week for 13 weeks, followed by a 13-week recovery period. Animals were euthanized following completion of exposure at 2, 6, or 13 weeks and at post exposure weeks 4 or 13. No mortality, overt signs of toxicity, body weight changes, haematological or clinical chemistry values were related to exposure.

At 50 ppm, relative liver weights were significantly increased in males but with no accompanying histopathological changes. Males at this exposure level also showed alterations in renal function during the study (reduced urine specific gravity and urine osmolality, changes in sodium and potassium excretion rates and increased urine volume) which were not present during the recovery period.

The only histopathological findings were in the kidney, in male rats only, particularly those exposed to 5.1 or 51 ppm. Hyaline droplets accumulated in the proximal convoluted tubule during the exposure period and resolved during the recovery period. Males at 5.1 and 51 ppm also had protein accumulation, tubular hyperplasia (regeneration), tubular proteinosis, interstitial nephritis and glomerular basement thickening. These changes did not resolve by the end of the recovery period and were also seen in some males in the control and 1 ppm groups; they are consistent with a male, rat-specific, glomerulonephropathy, which is seen spontaneously in older male rats.

This study indicates an overall low degree of systemic toxicity following subchronic inhalation exposure of dicyclopentadiene at exposure levels up to 50 ppm.