Registration Dossier

Administrative data

Description of key information

Oral and inhalation exposure to dicyclopentadiene resulted in nephrotoxicity (alterations in renal function and kidney morphology) in male rats only and characteristic of hyaline droplet nephropathy, which is not relevant for human risk assessment. Other changes which were seen in male rats in all groups (including controls) were characteristic of chronic glomerulonephropathy. Mortality was seen in rats dosed with 100 mg/kg/day orally and in mice exposed to 51 ppm by inhalation. Apart from the effects on the kidney (in male rats only), there were few histopathological changes. Single cell necrosis in the liver and fatty changes in the adrenal glands were seen in the oral rat study only.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
Combined repeated dose and reproduction / developmental screening Test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Not specified in the study report
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study, guideline study, available as unpublished report and summary information sheet (provided by Japan Chemical Industry Ecology-Toxicology and Information Center (JETOC)), minor restrictions in design and/or reporting but otherwise adequate for assessment.
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Zeon; Lot No. D93028
- Expiration date of the lot/batch: Not specified
- Purity test date: Not specified
- Storage condition of test material: Test substance was stored at room temperature
- Stability under test conditions: stable during the study period

FORM AS APPLIED IN THE TEST (if different from that of starting material) : colorless liquid with camphor-like odor

OTHER SPECIFICS:
purity 94.65%
Molecular weight: 132.22
Boling Point: 170°C
Melting Point: -1°C
Density: 0.986
Solubility in water: 40 ppm
Vapour pressure: 9.75 mm Hg (37.7°C)
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crj:CD
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan, Inc.
- Age at study initiation: 8 weeks
- Weight at study initiation: males 304-339 g, females 186-227 g
- Housing: individually, except during mating, in polycarbonate cages
- Diet: CRF-1 (Oriental Yeast Co) assumed ad libitum
- Water: ultraviolet irradiated water (assumed ad libitum)
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20-25°C
- Humidity: 40-70%
- Air changes: approximately 12 per hr
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: Not reported
Route of administration:
oral: gavage
Vehicle:
olive oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Test substance mixed with olive oil, dose rate 5 mL/Kg bodyweight

VEHICLE
- Justification for use and choice of vehicle (if other than water): Olive Oil
- Amount of vehicle (if gavage): 5 mL/Kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability and achieved concentration of dosing preparations was confirmed prior to dosing
Duration of treatment / exposure:
Males 44 days; Females from 14 days before mating through gestation and parturition until day 3 of lactation
Frequency of treatment:
once daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Basis: nominal in olive oil
Dose / conc.:
4 mg/kg bw/day (nominal)
Remarks:
Basis: nominal in olive oil
Dose / conc.:
20 mg/kg bw/day (nominal)
Remarks:
Basis: nominal in olive oil
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Basis: nominal in olive oil
No. of animals per sex per dose:
10/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: - Based on the results obtained in a 10 day oral dosing preliminary study, where doses of 0, 30, 100 and 300 mg/Kg were administered.
- Rationale for animal assignment (if not random): stratified random sampling method by weight
Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: Yes

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes (males only)
- Time schedule for collection of blood: termination
- Anaesthetic used for blood collection: Yes (sodium thiopental)
- Animals fasted: Yes (assumed)
- How many animals: 10/group
- Parameters examined: red blood cell, white blood cell, platelets, haemoglobin, haematocrit, differential white cell count, reticulocyte, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration

CLINICAL CHEMISTRY: Yes (males only)
- Time schedule for collection of blood: termination
- Anaesthetic used for blood collection: Yes (sodium thiopental)
- Animals fasted: Yes (assumed)
- How many animals: 10/group
- Parameters examined: GOT, GPT, ALP, ¿-GTP, urea nitrogen, glucose, total cholesterol, triglycerides, creatinine, total bilirubin, total protein, albumin, A/G ratio, calcium, inorganic phosphorus, sodium, potassium, chloride

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

ORGAN WEIGHTS: Yes
- organs weighed: thymus, liver, kidneys, adrenals, testes, epididymes

HISTOPATHOLOGY: Yes (liver, kidney and adrenals all groups, other tissues controls and 100 mg/kg groups only)
- tissues examined: thymus, liver, kidneys, adrenals, testes, epididymes, brain, heart, spleen, ovaries,
Statistics:
Bartlett's test if uniformly distributed analysis of variance, Kruskal-Wallis if non-uniform for quantitative data. When significant differences found between groups,Dunnett-type test or Scheff test. Significance level of 5% or less.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Two females in the high dose (100 mg/kg) group died. In these decedents the following major observations were noted: lung congestion, enlargement of the
adrenal gland, and bleeding of the gastric mucosa and thymus.

Transient salivation was observed immediately after dosing in the 100 mg/kg dose group after the start of dosing and was still observed after eight days in more
than half of male and female rats. This was also seen in a few male rats in the 20 and 4 mg/kg dose groups.
Mortality:
mortality observed, treatment-related
Description (incidence):
Two females in the high dose (100 mg/kg) group died. In these decedents the following major observations were noted: lung congestion, enlargement of the adrenal gland, and bleeding of the gastric mucosa and thymus.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Both male and female rats showed suppression of body weight gain and decrease in food consumption at 100 mg/Kg.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Both male and female rats showed suppression of body weight gain and decrease in food consumption at 100 mg/Kg.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Decrease in RBC count and hematocrit value in males in the 20 mg/Kg bw/day dose group were observed as was a decrease in hemoglobin concentration in males of the 100 mg/Kg bw/day dose
group. These were well recognized and within the range of values of both physiological changes. There were no hematological changes ascribable to the test material in any group.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Blood chemistry examinations in male rats in the 100 mg/Kg bw/day dose group showed increases in GOT and GPT.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
There were increased liver and kidney weights in male rats in the 100 mg/Kg bw/dose group.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Swelling of the liver; swollen adrenal glands; multifocal gray-white discoloration of the kidney were observed in the 100 mg/Kg bw/day male rats. Enlargement of the liver in one 20 mg/Kg bw/day male rats was also observed but determined to be incidental. Other observations included polycystic kidney enlargement, testicular atrophy, enlargement of the spleen, and subcutaneous nodules.

In the 2 females in the 100 mg/Kg bw/day dose group that died, the following major observations were noted: lung congestion, enlargement of the adrenal gland, and bleeding of the gastric mucosa and thymus.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In male rats given 100 mg/Kg, single cell necrosis in liver, and hyaline droplets and basophilic changes in tubular epithelium of kidneys was seen. Increase in fatty droplets in fascicular zone of adrenals was observed in both males and females in the 100 mg/Kg bw/day dose group. Similar histopathological changes were seen in kidneys of 4, 20 mg/Kg bw/day dose group male rats and in
adrenals of 20 mg/Kg bw/day dose group male rats.
Details on results:
CLINICAL SIGNS AND MORTALITY
- Two females in the high dose (100 mg/kg) group died. Transient salivation after dosing at 100 mg/kg for the initial 8 days of dosing was present in approximately half of the males and females. Also occasionally present in males at the two lower doses.

BODY WEIGHT AND WEIGHT GAIN
- Males and surviving females showed slight suppression of body wt gain.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
- Males and surviving females showed slightly decreased food consumption.

HAEMATOLOGY
- No treatment-related effects.

CLINICAL CHEMISTRY
- Blood chemistry of 100 mg/kg males showed increase in glutamic oxaloacetic transaminase (GOT) and glutamic-pyruvate transaminase (GPT).

ORGAN WEIGHTS
- Increased weight of liver and kidneys of male rats given 100 mg/kg (neither achieved statistical significance) and statistically significantly increased actual and relative liver weight in males at 20 mg/kg/day.

HISTOPATHOLOGY: NON-NEOPLASTIC
- In male rats given 100 mg/kg, single cell necrosis in liver, and hyaline droplets and basophilic changes in tubular epithelium of kidneys under microscopic examination were observed. Increase in fatty droplets in fascicular zone of adrenals was observed in both males and females in the 100 mg/kg group. Similar histopathological changes were seen in kidneys of four 20 mg/kg group male rats and in adrenals of 20 mg/kg group male rats.
Key result
Dose descriptor:
NOAEL
Effect level:
4 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Systemic Toxicity
Key result
Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Systemic Toxicity
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
20 mg/kg bw/day (nominal)
System:
immune system
Organ:
adrenal glands
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Table 1. Results of the Hematological Examination in Male Rats

Dose (mg/Kg/day)

0

4

20

100

Number of animals

10

10

10

10

RBC (104/µL)

887 ± 31.1

885 ± 22.7

836 ± 28.4**

849 ± 42.1*

Ht (PCV) (%)

47.7 ± 1.42

47.4 ± 1.55

45.9 ± 1.75*

45.9 ± 1.64*

Hb (g/dL)

16.1 ± 0.52

16.0 ± 0.57

15.5 ± 0.64

15.4 ± 0.62*

Reticulo (%)

26 ± 3.5

24 ± 2.6

27 ± 4.0

30 ± 5.3

MCV (µm3)

53.9 ± 1.04

53.6 ± 1.67

54.9 ± 1.55

54.1 ± 1.43

MCH (pg)

18.2 ± 0.51

18.1 ± 0.61

18.6 ± 0.64

18.2 ± 0.56

MCHC (%)

33.7 ± 0.58

33.8 ± 0.45

33.9 ± 0.40

33.6 ± 0.38

Plt (104/µL)

103.6 ± 14.82

101.3 ± 12.53

104.6 ± 16.55

110.7 ± 10.26

WBC (102/µL)

94 ± 27.8

98 ± 29.9

117 ± 27.6

114 ± 23.2

Differential Leukocyte Counts (%)

Lymphocytes

80 ± 5.4

81 ± 6.0

81 ± 8.1

78 ± 5.7

Neutrophils

 

 

 

 

   Segmented

10 ± 4.4

12 ± 4.4

12 ± 7.3

16 ± 5.8

   Band

0 ± 0.7

1 ± 0.8

0 ± 0.5

0 ± 0.5

Eosinophils

1 ± 1.1

1 ± 0.4

1 ± 1.1

1 ± 0.8

Basophils

0 ± 0.0

0 ± 0.0

0 ± 0.0

0 ± 0.0

Monocytes

9 ± 3.3

7 ± 3.6

6 ± 2.6

5 ± 2.9

Values are expressed as Mean ± S.D.

* Significantly different from the control group value at P<0.05

** Significantly different from the control group value at P<0.01

 

Table 2. Results of the Blood Chemistry Examination in Male Rats

Dose (mg/Kg/day)

0

4

20

100

Number of animals

10

10

10

10

GOT (AsT) (IU/L)

63 ± 1.1

70 ± 10.3

63 ± 10.8

88 ± 24.8

GPT (AlT) (IU/L)

24 ± 4.2

22 ± 4.1

23 ± 5.2

45 ± 16.5

¿-GPT (IU/L)

0 ± 0.0

0 ± 0.0

1 ± 2.2

1 ± 2.5

ALP (IU/L)

285 ± 55.6

270 ± 62.5

257 ± 42.5

297 ± 47.3

Total Bilirubin (mg/dL)

0.1 ± 0.07

0.1 ± 0.06

0.1 ± 0.08

0.1 ± 0.08

Urea Nitrogen (mg/dL)

15.3 ± 2.86

16.2 ± 2.44

18.0 ± 7.98

15.6 ± 1.70

Creatinine (mg/dL)

0.5 ± 0.07

0.5 ± 0.07

0.5 ± 0.08

0.5 ± 0.06

Glucose (mg/dL)

148 ± 32.9

141 ± 8.7

139 ± 18.3

133 ± 7.6

Total Chol. (mg/dL)

80 ± 12.7

73 ± 17.0

85 ± 27.3

101 ± 21.0

Triglyceride (mg/dL)

73 ± 32.0

65 ± 33.7

68 ± 25.3

63 ± 30.8

Total Protein (g/dL)

6.64 ± 0.266

6.40 ± 0.307

6.43 ± 0.254

6.66 ± 0.34

Albumin (g/dL)

3.91 ± 0.109

3.78 ± 0.145

3.79 ± 0.076

3.81 ± 0.14

A/G Ratio

1.44 ± 0.086

1.44 ± 0.080

1.45 ± 0.123

1.34 ± 0.08

Inorganic Phos. (mg/dL)

7.6 ± 0.40

7.8 ± 0.32

8.1 ± 0.57

8.0 ± 0.44

Ca (mg/dL)

9.5 ± 0.24

9.2 ± 0.21**

9.6 ± 0.26

9.7 ± 0.10

Na (mEq/L)

144 ± 1.3

144 ± 0.8

143 ± 1.2

144 ± 1.0

K (mEq/L)

4.5 ± 0.22

4.3 ± 0.16

4.6 ± 0.63

4.6 ± 0.18

Cl (mEq/L)

103 ± 1.5

103 ± 1.8

102 ± 1.6

101 ± 0.9

Values are expressed as Mean ± S.D.

** Significantly different from the control group value at P<0.01

 

Table 3. Absolute and Relative Organ Weights

Dose (mg/Kg/day)

0

4

20

100

Male Rats

Number of animals

10

10

10

10

Body weight (g)

494 ± 38.2

488 ± 26.0

487 ± 24.2

469 ± 29.2

Absolute Organ Weight

 

 

 

 

Thymus (mg)

310 ± 56.3

366 ± 79.1

348 ± 56.0

345 ± 56.0

Liver (g)

13.38 ± 1.434

13.04 ± 1.232

13.44 ± 0.851

15.27 ± 1.43

Kidneys (g)

2.85 ± 0.350

3.18 ± 0.251

3.42 ± 0.271*

3.33 ± 0.49

Adrenals (mg)

58.1 ± 6.37

59.8 ± 6.83

59.6 ± 7.32

61.5 ± 6.59

Testes (g)

3.12 ± 0.552

3.39 ± 0.287

3.05 ± 0.706

3.06 ± 0.67

Epididymides (g)

1.14 ± 0.167

1.17 ± 0.132

1.08 ± 0.222

1.10 ± 0.11

 

Relative Organ Weight

 

 

 

 

Thymus (mg%)

63 ± 12.1

76 ± 19.4

72 ± 11.3

74 ± 12.0

Liver (g%)

2.71 ± 0.141

2.67 ± 0.153

2.76 ± 0.149

3.25 ± 0.13

Kidneys (g%)

0.58 ± 0.057

0.65 ± 0.054

0.70 ± 0.058**

0.71 ± 0.08

Adrenals (mg%)

11.8 ± 1.17

12.3 ± 1.40

12.3 ± 1.39

13.2 ± 1.62

Testes (g%)

0.63 ± 0.103

0.70 ± 0.073

0.63 ± 0.146

0.66 ± 0.15

Epididymides (g%)

0.23 ± 0.035

0.24 ± 0.033

0.22 ± 0.048

0.23 ± 0.02

Female Rats

Number of animals

9

8

5

7

Body weight (g)

319 ± 23.6

314 ± 9.4

325 ± 24.3

298 ± 8.9

Absolute Organ Weight

 

 

 

 

Thymus (mg)

207 ± 48.8

223 ± 68.0

217 ± 62.4

205 ± 71.2

Liver (g)

13.46 ± 1.724

13.30 ± 1.640

13.56 ± 2.000

13.35 ± 1.77

Kidneys (g)

1.97 ± 0.201

1.89 ± 0.080

2.06 ± 0.201

1.96 ± 0.13

Adrenals (mg)

72.9 ± 7.17

71.0 ± 9.16

70.9 ± 3.86

71.3 ± 10.3

 

Relative Organ Weight

 

 

 

 

Thymus (mg%)

65 ± 16.6

71 ± 24.3

67 ± 18.3

69 ± 23.5

Liver (g%)

4.21 ± 0.326

4.22 ± 0.470

4.17 ± 0.353

4.48 ± 0.53

Kidneys (g%)

0.62 ± 0.030

0.60 ± 0.029

0.64 ± 0.017

0.66 ± 0.03

Adrenals (mg%)

22.9 ± 2.29

22.6 ± 2.95

22.0 ± 2.37

23.9 ± 3.24

Values are expressed as Mean ± S.D.

* Significantly different from the control group value at P<0.05

** Significantly different from the control group value at P<0.01

 

Table 4. Summary of Necropsy Findings

Sex

Males

Females

Dose

(mg/Kg /day)

0

4

20

100

0

4

20

100

Number of animals

10

10

10

10

10

10

10

8

Organ Findings

Liver

 

Enlargement

0

0

1

3

0

0

0

0

Kidneys

 

Discoloration

0

0

0

1

0

0

0

0

Whitish Dots

0

0

0

1

0

0

0

0

Enlargement

0

0

1

0

0

0

0

0

Multiple Cysts

0

0

1

0

0

0

0

0

Testes

 

Atrophy

1

0

2

1

 

 

 

 

Adrenals

 

Enlargement

0

0

0

1

0

0

0

0

Spleen

 

Enlargement

0

0

1

0

0

0

0

0

Subcutis

 

Nodule

0

0

0

0

0

0

1

0

 

Table 5. Summary of Histopathological Findings

Sex

Males

Females

Dose

(mg/Kg /day)

0

4

20

100

0

4

20

100

Number of animals

10

10

10

10

10

10

10

8

Organ Findings

Liver

 

Single cell necrosis

0

0

0

7

0

*

*

ND

Focal necrosis

0

0

0

0

1

*

*

ND

Multilocular biliary cyst

0

0

1

0

0

*

*

ND

Kidneys

 

Increase of hyaline droplets in the tubular epithelium

0

10

10

10

0

*

*

ND

Basophilic changes of the tubular epithelium

0

0

3

2

0

*

*

ND

Cystic Dilatation of renal tubules

1

0

0

0

0

*

*

ND

Multiple Cyst

0

0

1

0

0

*

*

ND

Testes

 

Atrophy of seminiferous tubules

1

*

2/2#

1

 

 

 

 

Adrenals

 

Increase of fatty droplets in the fascicular zone

0

0

3

8

0

0

0

 

Spleen

 

Hemosiderosis

0

*

*

0

1

*

*

ND

Subcutis

 

Mammary adenoma

*

*

*

*

*

*

1/1#

ND

# Number of animals showing lesion / Number of animals examined

* Not examined

ND – Value not determinable due to unclear presentation in study report table.

Conclusions:
Dicyclopentadiene induced systemic toxicity in male and female rats including death of two females at the 100 mg/kg/day dose level. The NOAEL for systemic effects in female rats was determined to be 20 mg/Kg/day. The NOAEL for systemic effects in male rats was determined to be 4 mg/Kg/day.
Executive summary:

In a combined repeat dose toxicity study with reproduction/developmental toxicity screening, groups of 10 males and 10 females were dosed by oral gavage with solutions of 0, 4, 20 or 100 mg/Kg/day DCPD in olive oil. Animals were dosed for 2 weeks prior to mating and during mating (approximately 2 weeks). Males and females were then dosed through gestation until day 3 of lactation. Females were killed on day 4 of lactation and males were killed on day 45 of the study.

Two females at 100 mg/kg/day died during the study and surviving males and females showed decreased food consumption and bodyweight gain at this dose level. Pathological changes in the liver and kidney were seen in males dosed at 100 mg/Kg/day (single cell necrosis in the liver, hyaline droplet formation and basophilic changes in the tubular epithelium of the kidney) and an increase in fatty droplets in the adrenals was observed in both males and females in the 100 mg/Kg group. Similar changes were seen in the kidney and adrenals of some male rats dosed at 20 mg/Kg group male rats.

The NOAEL for systemic toxicity was therefore considered to be 20 mg/Kg/day for females and 4 mg/Kg/day for male rats.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
4 mg/kg bw/day
Study duration:
subacute
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
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: animal experimental study, also published in peer-reviewed literature, minor restrictions in design and reporting but otherwise acceptable for assessment.
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Version / remarks:
not specified
GLP compliance:
no
Remarks:
Klimisch Cat 2
Limit test:
no
Species:
mouse
Strain:
other: B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratory, Portage, MI, USA
- Age on receipt: 30-34 days
- Health assessment: confirmed following arrival
- Weight at study initiation: no data
- Housing: individually in stainless steel wire mesh suspended cages
- Diet: powdered NIH-07 diet ad libitum except during exposure
- Water: ad libitum except during exposure
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature: 68-72°F non-exposure period
- Humidity: 40-60% non-exposure period
- Air changes (per hr): no data
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: July 1981 - January 1981
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: air
Remarks on MMAD:
MMAD / GSD: Not applicable
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Stainless steel rectangular (2mx2mx1m) exposure chamber with glass windows and door in front wall (total volume 4350 L).
- Method of holding animals in test chamber: individually in suspended stainless steel wire mesh cage with stainless steel pans between each layer of cages to prevent contamination. Cage positions were rotated routinely.
- System of generating particulates/aerosols: DCPD vapour was generated by heating the liquid in a Pyrex tube using a minimum amount of heat to prevent decomposition and formation of CPD. Filtered air was used to dilute the vapour prior to introduction into the chamber.
- Temperature and humidity in air chamber: 70-79°F, 39-68%
- Air flow rate: 2000 L/min

TEST ATMOSPHERE
- Brief description of analytical method used: Chamber concentrations were analysed at hourly intervals by gas chromatography/flame ionization detection.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
13 wks
Frequency of treatment:
6 hr/day, 5 days/wk
Remarks:
Doses / Concentrations:
0, 1, 5, 50 ppm
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
1, 5.1, 51 ppm
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
0, 5.4, 27.6, 276 mg/m3
Basis:
analytical conc.
No. of animals per sex per dose:
45
Control animals:
yes
Details on study design:
Post-exposure observation periods of 4 and 13 weeks.
9 mice/sex/dose were scheduled for sacrifice after 2, 6 and 13 weeks of exposure and 4 and 13 weeks post-exposure.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- During exposure mice were observed several times through the chamber window.

DETAILED CLINICAL OBSERVATIONS: Yes
- Mice were observed for clinical signs before and after each exposure and daily during the recovery period.

BODY WEIGHT: Yes
- Recorded at study initiation, weekly during both the exposure period and the first 5 weeks of the recovery period, and then every 2 weeks. Animals were also weighed before termination.

FOOD CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- High dose mice received ophthalmoscopic examination before sacrifice

HAEMATOLOGY: Yes
- Haematology analyses were performed on all mice prior to sacrifice after 2, 6 and 13 week exposure and 4 and 13 week post-exposure with blood from the orbital sinus. Erythrocyte count, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin and concentration, and total/differential white blood cell counts were determined.

CLINICAL CHEMISTRY: Yes
- Serum chemistry analyses were performed on all mice prior to sacrifice after 2, 6 and 13 week exposure and 4 and 13 week post-exposure with blood from the orbital sinus. Serum was analyzed for creatinine, urea nitrogen, calcium, phosphrous, chloride, alanine aminotransferase, aspartate aminotransferase, total protein, albumin, total bilirubin, alkaline phosphatase, glucose and osmolality.

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Necropsies were conducted on all mice.
- Kidneys, lungs, liver and testes were weighed.
- Adrenals, bone and bone marrow (sternum), brain, 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 were preserved for microscopic evaluation.

HISTOPATHOLOGY: Yes
- Organs were examined microscopically in control and high dose mice sacrificed after 13 weeks of exposure.
Statistics:
Analysis of variance, Bartlett’s test, Duncan’s multiple range test, F-test, Student’s t-test, Cochran t-test (applied when appropriate).
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):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
- Ten males and 9 female mice exposed to 51 ppm DCPD died during the study; no more than 2 mice died at any other level.
- No significant clinical signs or body weight changes were noted prior to death. The likely cause of death appeared to be pulmonary congestion and possibly renal failure. These effects were not seen in mice sacrificed at the end of the study.
- During exposure, a few of the mice at 51 and 5.1 ppm showed coordination loss and/or decreased activity.

BODY WEIGHT AND WEIGHT GAIN
- Males and females in the 51 ppm group showed significant elevation in body wt gain that returned to parity with control values during recovery.
Dose descriptor:
NOAEC
Effect level:
5 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: 27.6 mg/m3. Mortality (20%) occurred in the high-dose mice during the study
Critical effects observed:
not specified

A number of statistically significant alterations were noted in this study but the aetiology and association with dicyclopentadiene exposure are unclear. Approximately 20 percent of mice exposed to 51 ppm died during the exposure regimen. The cause of death was pulmonary congestion yet similar lung lesions were not found in animals terminated during the study. Also, female mice exposed to 51 ppm showed an increase in body weight during the last few weeks. A potential effect of dicyclopentadiene was seen in the female mice given 64 exposures to 51 or 5.1 ppm was a decrease in serum albumin indicative of slight liver dysfunction (7% difference from control); absolute and relative liver weights were also increased. No morphological changes were found to indicate any effect of dicyclopentadiene exposure. Thus any effect of dicyclopentadiene on the livers of female mice was considered to be minimal in severity.

Conclusions:
Although there were no overt signs of toxicity due to dicylopentadiene, approximately 20% of mice died primarily as a result of pulmonary congestion. The aetiology and association with dicyclopentadiene exposure are unclear. The NOAEC is concluded to be 5.1 ppm (27.6 mg/m3).
Executive summary:

Groups of 45 male and 45 female B6C3F1 mice were exposed by inhalation, 6 hr/day, 5 days/week, for 13 weeks (64 exposures) to dicyclopentadiene vapour at concentrations of 0 (air control), 1, 5.1 or 51 ppm (analysed concentrations). Animals were sacrificed after 10, 30 and 64 inhalation exposures and post-exposure sacrifices were made at 29 and 92 days following the last exposure. Clinical observations, body weights, blood clinical chemistry and haematology, ophthalmology, organ weights and histopathology evaluations were made during the study. A number of statistically significant alterations were noted in this study but the aetiology and association with dicyclopentadiene exposure are unclear. There were no overt signs of toxicity although approximately 20% of the mice of the 51 ppm exposure group died during the exposure period, primarily due to pulmonary congestion. The NOAEC is concluded to be 5.1 ppm (27.6 mg/m3).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
27.6 mg/m³
Study duration:
subchronic
Species:
mouse

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
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: animal experimental study, also published in peer-reviewed literature, minor restrictions in design and reporting but otherwise acceptable for assessment.
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Version / remarks:
not specified
GLP compliance:
no
Remarks:
Klimisch Cat 2
Limit test:
no
Species:
mouse
Strain:
other: B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratory, Portage, MI, USA
- Age on receipt: 30-34 days
- Health assessment: confirmed following arrival
- Weight at study initiation: no data
- Housing: individually in stainless steel wire mesh suspended cages
- Diet: powdered NIH-07 diet ad libitum except during exposure
- Water: ad libitum except during exposure
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature: 68-72°F non-exposure period
- Humidity: 40-60% non-exposure period
- Air changes (per hr): no data
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: July 1981 - January 1981
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: air
Remarks on MMAD:
MMAD / GSD: Not applicable
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Stainless steel rectangular (2mx2mx1m) exposure chamber with glass windows and door in front wall (total volume 4350 L).
- Method of holding animals in test chamber: individually in suspended stainless steel wire mesh cage with stainless steel pans between each layer of cages to prevent contamination. Cage positions were rotated routinely.
- System of generating particulates/aerosols: DCPD vapour was generated by heating the liquid in a Pyrex tube using a minimum amount of heat to prevent decomposition and formation of CPD. Filtered air was used to dilute the vapour prior to introduction into the chamber.
- Temperature and humidity in air chamber: 70-79°F, 39-68%
- Air flow rate: 2000 L/min

TEST ATMOSPHERE
- Brief description of analytical method used: Chamber concentrations were analysed at hourly intervals by gas chromatography/flame ionization detection.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
13 wks
Frequency of treatment:
6 hr/day, 5 days/wk
Remarks:
Doses / Concentrations:
0, 1, 5, 50 ppm
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
1, 5.1, 51 ppm
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
0, 5.4, 27.6, 276 mg/m3
Basis:
analytical conc.
No. of animals per sex per dose:
45
Control animals:
yes
Details on study design:
Post-exposure observation periods of 4 and 13 weeks.
9 mice/sex/dose were scheduled for sacrifice after 2, 6 and 13 weeks of exposure and 4 and 13 weeks post-exposure.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- During exposure mice were observed several times through the chamber window.

DETAILED CLINICAL OBSERVATIONS: Yes
- Mice were observed for clinical signs before and after each exposure and daily during the recovery period.

BODY WEIGHT: Yes
- Recorded at study initiation, weekly during both the exposure period and the first 5 weeks of the recovery period, and then every 2 weeks. Animals were also weighed before termination.

FOOD CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- High dose mice received ophthalmoscopic examination before sacrifice

HAEMATOLOGY: Yes
- Haematology analyses were performed on all mice prior to sacrifice after 2, 6 and 13 week exposure and 4 and 13 week post-exposure with blood from the orbital sinus. Erythrocyte count, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin and concentration, and total/differential white blood cell counts were determined.

CLINICAL CHEMISTRY: Yes
- Serum chemistry analyses were performed on all mice prior to sacrifice after 2, 6 and 13 week exposure and 4 and 13 week post-exposure with blood from the orbital sinus. Serum was analyzed for creatinine, urea nitrogen, calcium, phosphrous, chloride, alanine aminotransferase, aspartate aminotransferase, total protein, albumin, total bilirubin, alkaline phosphatase, glucose and osmolality.

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Necropsies were conducted on all mice.
- Kidneys, lungs, liver and testes were weighed.
- Adrenals, bone and bone marrow (sternum), brain, 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 were preserved for microscopic evaluation.

HISTOPATHOLOGY: Yes
- Organs were examined microscopically in control and high dose mice sacrificed after 13 weeks of exposure.
Statistics:
Analysis of variance, Bartlett’s test, Duncan’s multiple range test, F-test, Student’s t-test, Cochran t-test (applied when appropriate).
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):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
- Ten males and 9 female mice exposed to 51 ppm DCPD died during the study; no more than 2 mice died at any other level.
- No significant clinical signs or body weight changes were noted prior to death. The likely cause of death appeared to be pulmonary congestion and possibly renal failure. These effects were not seen in mice sacrificed at the end of the study.
- During exposure, a few of the mice at 51 and 5.1 ppm showed coordination loss and/or decreased activity.

BODY WEIGHT AND WEIGHT GAIN
- Males and females in the 51 ppm group showed significant elevation in body wt gain that returned to parity with control values during recovery.
Dose descriptor:
NOAEC
Effect level:
5 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: 27.6 mg/m3. Mortality (20%) occurred in the high-dose mice during the study
Critical effects observed:
not specified

A number of statistically significant alterations were noted in this study but the aetiology and association with dicyclopentadiene exposure are unclear. Approximately 20 percent of mice exposed to 51 ppm died during the exposure regimen. The cause of death was pulmonary congestion yet similar lung lesions were not found in animals terminated during the study. Also, female mice exposed to 51 ppm showed an increase in body weight during the last few weeks. A potential effect of dicyclopentadiene was seen in the female mice given 64 exposures to 51 or 5.1 ppm was a decrease in serum albumin indicative of slight liver dysfunction (7% difference from control); absolute and relative liver weights were also increased. No morphological changes were found to indicate any effect of dicyclopentadiene exposure. Thus any effect of dicyclopentadiene on the livers of female mice was considered to be minimal in severity.

Conclusions:
Although there were no overt signs of toxicity due to dicylopentadiene, approximately 20% of mice died primarily as a result of pulmonary congestion. The aetiology and association with dicyclopentadiene exposure are unclear. The NOAEC is concluded to be 5.1 ppm (27.6 mg/m3).
Executive summary:

Groups of 45 male and 45 female B6C3F1 mice were exposed by inhalation, 6 hr/day, 5 days/week, for 13 weeks (64 exposures) to dicyclopentadiene vapour at concentrations of 0 (air control), 1, 5.1 or 51 ppm (analysed concentrations). Animals were sacrificed after 10, 30 and 64 inhalation exposures and post-exposure sacrifices were made at 29 and 92 days following the last exposure. Clinical observations, body weights, blood clinical chemistry and haematology, ophthalmology, organ weights and histopathology evaluations were made during the study. A number of statistically significant alterations were noted in this study but the aetiology and association with dicyclopentadiene exposure are unclear. There were no overt signs of toxicity although approximately 20% of the mice of the 51 ppm exposure group died during the exposure period, primarily due to pulmonary congestion. The NOAEC is concluded to be 5.1 ppm (27.6 mg/m3).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
27.6 mg/m³
Study duration:
subchronic
Species:
mouse

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Non human Information

Oral 

The key study is considered to be a combined repeat dose toxicity study with reproduction/developmental toxicity screening (JETOC, 1998a). Systemic toxicity at 100 mg/kg/day was indicated by the mortality of two female rats and, decreased food consumption and bodyweight gain in both males and females. Pathological changes in the liver (single cell necrosis, males only) and adrenals (increase in fatty droplets, males and females). Similar changes were seen in the adrenals of some male rats dosed at 20 mg/kg/day. Male rat specific kidney pathology (hyaline droplet formation and basophilic changes in the tubular epithelium of the kidney) was observed in males in all dose groups. These findings are not relevant for human risk assessment.

The no effect level for systemic toxicity was therefore considered to be 20 mg/kg/day for females and 4 mg/kg/day for male rats (based on the adrenal effects at higher dose levels).

In the supporting study (Litton Bionetics, 1980), dicyclopentadiene was administered by incorporation into the diet at concentrations of 100, 300 and 1000 ppm to male and female beagle dogs for 13 weeks. No significant toxicity was seen at any dose level. Minor indications of intestinal distress (vomiting and soft stools) seen at the highest dose (1000 ppm) were considered to be of no toxicological significance. The NOAEL of dicyclopentadiene was therefore considered to be 1000 ppm (25 mg/kg/day) to beagle dogs following 13 week dietary administration.

Inhalation

Key studies were conducted in Fischer 344 rats and B6C3F1 mice (Bushy Run, 1982). Vapour generation was carefully controlled and analysed to ensure that no cyclopentadiene was formed during vapour generation. Animals were exposed (whole body) to vapours of 0, 1, 5 or 50 ppm dicyclopentadiene, 6 hr/day, 5 days/week for 13 weeks, followed by a 13-week recovery period. Animals were killed following completion of exposure at 2, 6, or 13 weeks and at post exposure weeks 4 or 13. 

There was no mortality in any of the rats but approximately 20% of the mice (both sexes) of the 51 ppm exposure group died during the exposure period.

In rats exposed to 51 ppm, relative liver weights were significantly increased in males.

The kidney effects seen only in male rats exposed to dicyclopentadiene were reported by Bevan et al (1992) to be characteristic of alpha2u-nephropathy, which is considered to be of no relevance to human risk assessment.

The rat study indicates an overall low degree of systemic toxicity following sub-chronic inhalation exposure of dicyclopentadiene at exposure levels up to 51 ppm.  As concluded by Bevan et al. (1992), increased liver weights in the absence of histopathologic changes were not considered to be an adverse effect. Excluding the male, rat-specific hyaline droplet nephropathy seen at 5.1 and 51 ppm during the exposure period, the no observed adverse effect concentration (NOAEC) in this study is therefore considered to be 51 ppm (276 mg/m3) for both male and female rats.  Since approximately 20% of the mice (both sexes) of the 51 ppm exposure group died during the exposure period, the NOAEC is concluded to be 5.1 ppm (27.6 mg/m3) for male and female mice.

Human studies

In studies in human volunteers to ascertain the odour threshold of dicyclopentadiene vapour for man and to determine the human sensory response (Kinkead et al, 1981), some inadvertent exposure to dicyclopentadiene vapour occurred during the 5 month investigation period. Three workers experienced transitory headaches during the first 2 months, but not during the last 3 months.


Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
Mortality in male and female mice at 276mg/m3 was associated with pulmonary congestion resulting from chronic irritation of the lung.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
Mortality in male and female mice at 276mg/m3 was associated with pulmonary congestion resulting from chronic irritation of the lung.

Repeated dose toxicity: via oral route - systemic effects (target organ) glandular: adrenal gland

Justification for classification or non-classification

Dicyclopentadiene is not classified for repeat dose toxicity under CLP.

While some effects were seen in repeat dose studies, these are believed to be due to chronic irritation of the lungs and gastrointestinal tract with secondary stress-related effects rather specific target organ toxicity. DCPD is irritating; therefore it is biologically plausible that the mortality is mostly attributable to respiratory and gastric irritation effects rather than systemic effects. Liver effects seen in the rat oral study were not considered sufficient to warrant classification.

Repeated exposure inhalation studies in rodents show mortality at levels that would be predicted by their LC50s, and appears related to respiratory irritation/portal-of-entry effects. The original Bushy Run 90-day study showed mortality in the mice at 276 mg/m3. The LC50 in mice is about half that of rats, which might be expected for a respiratory irritant (higher ventilation rate in the mouse). This justification for non-classification for the repeat dose toxicity (mortality in mice) is supported by the existing acute inhalation classification (Acute Cat 2).