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Repeated dose toxicity: oral

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Administrative data

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GLP study, guideline study, available as unpublished report and summary information sheet (provided by Japan Chemical Industry Ecology-Toxicology and Information Center (JETOC)), minor restrictions in design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.
Cross-reference
Reason / purpose:
reference to same study

Data source

Referenceopen allclose all

Reference Type:
other: Information sheet
Title:
Unnamed
Year:
1998
Report Date:
1998
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report Date:
1993

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): dicyclopentadiene
- Analytical purity: 94.65%
- Physical state: colourless liquid with a camphor-like odour
- Lot/batch No.: D93028
- Stability under test conditions: confirmed to be stable by the manufacturer for the study period
- Storage condition of test material: room temperature
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)

Test animals

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

Administration / exposure

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
Doses / concentrationsopen allclose all
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

Examinations

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.

Results and discussion

Results of examinations

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.

Effect levels

open allclose all
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

Target system / organ 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

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.