Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

There are no human data available for effects of dicyclopentadiene on fertility.

A key three generation reproductive toxicity study (dietary route of exposure, Klimisch 2, pre-GLP) conducted in Sprague-Dawley rats is available for assessment. Supportive data from a GLP OECD test guideline 422 study with Sprague Dawley rats is also available. Although the GLP report is written in Japanese, the study details were summarized in English in JETOC (1998a), and an abridged English translation of the GLP report is also available. In this study rats were dosed by oral gavage. Additionally, data from a Continuous Breeding Study in the rat (gavage, Klimisch 4) is also available.

 

There are no reproduction studies available using the dermal or inhalation routes of exposure to dicyclopentadiene.

Link to relevant study records

Referenceopen allclose all

Endpoint:
three-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1977-05-22 to 1979-07-30
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Non-guideline study, pre-GLP 1979 study, limitations in design and/or reporting but otherwise adequate for assessment
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
yes
Remarks:
There were only 2 dietary concentrations tested
GLP compliance:
no
Remarks:
study report is dated July 1979, pre-GLP
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: MC/B (2909 Highland Avenue, Norwood, Ohio 45212); Catalog No. TX 310
- Expiration date of the lot/batch: Not specified
- Purity test date: Analysis of DCPD was performed with a UC-W98 column post receipt on August 18, 1976. (Purity: 98 to 99%)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Closed container in the freezer
- Stability under test conditions: Stability analysis revealed that in the closed containers, the concentration dropped 27.6% for the 80 ppm level and 30.8% for the 750 ppm level over a 10-day period.
- Solubility and stability of the test substance in the solvent/vehicle: Because of poor water solubility, DCPD was prepared for administration to animals by dissolving it in corn oil (Mazola).

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: waxy solid converted to an easily measured liquid by slight warming

FORM AS APPLIED IN THE TEST (if different from that of starting material) : waxy solid converted to an easily measured liqud by slight warming

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
CRL:COB (SD) BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc. (Portage, Michigan, USA)
- Females (if applicable) nulliparous and non-pregnant: [yes/no]
- Age at study initiation: (P) - Weanling albino rats
- Weight at study initiation: Not specified
- Fasting period before study: Not specified
- Housing: housed individually (except when mating) in shoe box cages on AB-SORB-DRI bedding
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet (e.g. ad libitum): Purina Laboratory Chow meal provided ad libitum
- Water (e.g. ad libitum): water provided ad libitum
- Acclimation period: Acclimated to laboratory conditions for 11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Not specified
- Humidity (%): Not specified
- Air changes (per hr): Not specified
- Photoperiod (hrs dark / hrs light): Not specified

IN-LIFE DATES: From: 1977-05-22 To: Not specified
Route of administration:
oral: feed
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): Fresh diets were prepared weekly
- Mixing appropriate amounts with (Type of food): Diets prepared by adding the appropriate quantity of DCPD, dissolved in 300 mL of corn oil, to 10 Kg of Purina Laboratory Chow meal, and mixing for at least 15 minutes in a twin shell blender. Control diet was mixed with corn oil in the same fashion.
- Storage temperature of food: Stored at ambient conditions

VEHICLE
- Justification for use and choice of vehicle (if other than water): Because of poor water solubility, DCPD was prepared for administration to animals by dissolving it in corn oil (Mazola). The handling of DCPD itself was facilitated by slight warming, which converted the waxy solid to an easily measured liquid.
- Concentration in vehicle: at concentrations appropriate to the various studies
Details on mating procedure:
- M/F ratio per cage: Each male caged with two females of its dose group
- Length of cohabitation: 2 weeks
- Proof of pregnancy: Not specified
- Further matings after two unsuccessful attempts: Not specified
- After successful mating each pregnant female was caged (how): rats were returned to their respective cages and the females were allowed to litter
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Because of the possible loss from the diet through volatility of DCPD, samples of each week's dietary batch were analyzed by the LBI Chemistry Department. The analytical method employed gas-liquid chromatography and was supplied by the sponsor and developed at LBI.
Duration of treatment / exposure:
For 7 weeks prior to mating of the F0 parents through to study termination.
Frequency of treatment:
Continuous in diet
Details on study schedule:
- F0 rats were mated seven weeks after initiation of treated diet. One week after weaning the first litters (F1a pups), the F0 parents were remated, each male with a different pair of females from that of the first mating. One week after weaning the second litters (F1b pups), parent F0 rats were killed.
- Selected F1b pups were designated F1 parents and were approx. 100 days old when mated to produce the F2a litters and subsequently the F2b litters. Selected F2b pups were designated F2 parents and similarly used to produce the F3 a and b litters.
Dose / conc.:
0 ppm
Remarks:
Basis: nominal in diet
Group 1 (Control)
Dose / conc.:
80 ppm
Remarks:
Basis: nominal in diet
69.3 ppm (analytical concentration)
Group 2
Dose / conc.:
750 ppm
Remarks:
Basis: nominal in diet
693 ppm (analytical concentration)
Group 3
No. of animals per sex per dose:
10 males, 20 females
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: A clear rationale for selection of the doses that were used in the study is not provided in the study report. However, in a rat teratology study with DCPD conducted by the same laboratory in 1977 (LBI Project number 10734-05), the study report mentions that the dose levels used in the study were approved by Dr. E. Ross Hart of LBI based on previous studies.
- Rationale for animal assignment (if not random): Rats were assigned randomly to the study groups
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily observations were made of parent rats for mortality and general condition

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: At 4 and at 8-9 weeks, and shortly before each mating, parent rats were weighed

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes; At 4 and at 8-9 weeks, and shortly before each mating, the food consumption of parent rats was estimated
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
Oestrous cyclicity (parental animals):
No
Sperm parameters (parental animals):
No
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- At Day 4 each litter was reduced to eight total pups, four per sex if possible

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring:
- Gross abnormalities of pups
- Numbers of live and dead pups, and their mean body weight by sex at birth
- Number per sex Day 4 of lactation
- Number per sex and body weights Day 21 of lactation (weaning)

GROSS EXAMINATION OF DEAD PUPS: Yes; At weaning, gross necropsies were performed on approximately one-third of the first litters from all three generations, and on one-third of the F3b litters.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals (A gross necropsy was performed on each Parent F0 animal one week after weaning the second litters (F1b pups).
- Maternal animals: All surviving animals (A gross necropsy was performed on each Parent F0 animal one week after weaning the second litters (F1b pups).

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
No data
Postmortem examinations (offspring):
At weaning, gross necropsies were performed on approximately one-third of the first litters from all three generations, and on one-third of the F3b litters.
Statistics:
Student's t-test; Chi-square test
Reproductive indices:
Male and female fertility; gestation index.
Offspring viability indices:
Newborn viability; pup viability (Days 0-4); lactation viability (days 4-21); sex ratio Day 0; Live pups per litter
Clinical signs:
no effects observed
Description (incidence and severity):
One F0 female (No. 5196, 80 ppm) was found dead at Week 28, but all other F0 rats survived their portion of the study in generally good condition. Data for compound-treated groups were entirely comparable to control figures at each interval.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One F0 female (No. 5196, 80 ppm) was found dead at Week 28, but all other F0 rats survived their portion of the study in generally good condition. Data for compound-treated groups were entirely comparable to control figures at each interval.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
F0 parents - Data for compound-treated groups were entirely comparable to control figures at each interval.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
F0 - Data for compound-treated groups were entirely comparable to control figures at each interval.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
Reproductive and litter indices were unaffected by exposure to DCPD (please see Tables 3 and 4) below.
Key result
Dose descriptor:
NOAEL
Remarks:
F0 Parents
Effect level:
750 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Systemic Toxicity
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
F1b Parents - No clinical signs reported.
Mortality:
no mortality observed
Description (incidence):
F1b Parents - No mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
F1b Parents - At all intervals, rats in the compound-treated groups weighed as much as or more than the controls, except for the 80-ppm females at 20 weeks (just prior to the second mating). In this instance, the slightly lower mean body weight was not statistically significant.

F2b Parents - No meaningful differences between groups at the various intervals were seen with respect to body weight.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
F1b Parents - food consumption means were comparable among the groups, except that in both males and females of the 750-ppm group, the reductions at 20 weeks in food intake were statistically significant (p<0.05, Student's t-test).

F2b Parents - No meaningful differences between groups at the various intervals were seen with respect to food consumption.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
F1b Parents - At necropsy, no gross lesions were found in the F1b parent rats.

F2b Parents - Necropsy findings of the F2b parents were unexceptional.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
F1b Parents - In the first mating to produce the F2a generation, except for reduced female fertility in the 750-ppm group, litter data in all groups were comparable.
However, the 70% figure (Table 5) for the F2a was not statistically significantly different (Chisquare test) from the control index (95%). In addition, it may be noted that male No. 7349 in the 750-ppm group failed to sire a litter in either mating, and could thus be responsible for the lack of litters from two of six non-productive females at the first mating. It was therefore concluded that the apparently lowered fertility of the high-dose females at the first mating was not related to compound administration.

In the second mating to produce the F2b generation, fertility in the 750-ppm females was reduced. However, the 85% figure (Table 6) for the F2b's was not statistically significantly different (Chisquare test) from the control index (95%). In addition, it may be noted that male No. 7349 in the 750-ppm group failed to sire a litter in either mating, and could thus be responsible for the lack of litters from two of three non-productive females at the second mating. It was therefore concluded that the apparently lowered fertility of the high-dose females at the second mating is not related to compound administration.

F2b Parents - Although female fertility was only 80% and 83% in the DCPD-treated groups, control fertility was worse, only 65%. There was nothing in these records to suggest a compound-related effect.
Key result
Dose descriptor:
NOAEL
Remarks:
F1b Parents
Effect level:
80 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Systemic Toxicity
Key result
Dose descriptor:
NOAEL
Remarks:
F2b Parents
Effect level:
750 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Systemic toxicity
Key result
Critical effects observed:
no
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There was nothing in the data to distinguish DCPD-treated groups (F1a) from control rats. Observations of the pups did not indicate anything of importance in the three groups. One F1a pup in a 80-ppm litter had an opaque left eye, and one F1a pup in a 750-ppm litter had a crooked tail. Such isolated findings were not considered to be meaningful.

All F1b generation groups were comparable to one another with respect to both litter data and pup observations. A single instance of a pup in the 80-ppm group with an abnormality (a deformed hind foot) was not considered to be meaningful.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
No mortality observed in F1a offspring.

F1b Offspring - Group 1 (Control (0 ppm)): 3 males and 3 females were found dead on lactation days 0-6; Group 2 (80 ppm): 3 males and 3 females were found dead on lactation days 0-4; Group 3 (750 ppm): 2 males were found dead on lacation days 4-11; and one pup was found dead, partially cannabalized on lacation day 0. Please see table Table 7 (pup observations (F1b)) of the attached study report for further details.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
F1a Offspring - No compound-related findings were reported

F1b Offspring - No compound-related findings were reported
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
F1a Offspring - No compound-related findings were reported

F1b Offspring - No compound-related findings were reported
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
F1a Offspring - No compound-related findings were reported

F1b Offspring - No compound-related findings were reported
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
F1a Offspring - All groups were comparable to one another with respect to both litter data and pup observations.

F1b Offspring - All groups were comparable to one another with respect to both litter data and pup observations.
Key result
Dose descriptor:
NOAEL
Generation:
F1a
Effect level:
750 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reproductive Toxicity
Key result
Dose descriptor:
NOAEL
Generation:
F1b
Effect level:
750 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reproductive Toxicity
Key result
Critical effects observed:
no
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
F2a Offspring - No general findings of significance were recorded, but one male pup in the 80-ppm group was found to have had hydrocephalus.
F2b Offspring - General pup observations were unexceptional.

F3a Offspring - Pup general observations showed nothing to indicate compound-related effects
F3b Offspring - Pup general observations showed nothing to indicate compound-related effects
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
F2a Offspring - Group 1 (Control (0 ppm)): 1 male was found dead on lactation day 4; Group 2 (80 ppm): 2 females, 1 found dead and 1 in moribund condition (killed)) were found on lactation day 4 while 1 male was found dead on lacation day 20; Group 3 (750 ppm): 1 female was found dead on lactation day 4. Please see Table 12 (pup observations F2a in the attached study report for further details).

F2b Offspring - Group 1 (Control (0 ppm)): 1 male was found dead on lactation day 4; Group 2 (80 ppm): 2 males, 1 found dead on lactation day 2 and 1 found dead on lactation day 4; Group 3 (750 ppm): 1 male was found dead on lactation day 4. Please see Table 15 (pup observations F2b in the attached study report for further details).

F3a Offspring - Group 1 (Control (0 ppm)): Three males and 2 females found dead while 1 pup was cannibalized on lactation day 11. One male was found dead on lactation day 12; Group 2 (80 ppm): No mortality; Group 3 (750 ppm): 1 female found dead on lactation day 3 and 1 male found dead on lactation day 4.

F3b Offspring - Group 1 (Control (0 ppm)): 1 female was found dead and 3 females were cannabilized on lactation day 4; Group 2 (80 ppm): No mortality; Group 3 (750 ppm): 3 females were cannibalized on lactation day 4.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
F2a Offspring - No effect on body weights of F2a offspring was reported.
F2b Offspring - No effect on body weights of F2b offspring was reported.

F3a Offspring - No effect on body weights of F3a offspring was reported.
F3b Offspring - A slight reduction in mean pup weight at weaning (compared to controls) was noted in each compound-treated group, that for the high dose female pups being statistically significant (p<0.05; Student's t-test). The low-dose female pup weight, while numerically the same as that of the 750-ppm weanlings, was not statistically significant. Since mean weanling pup weights in the other generations were not appreciably different among the groups involved, the study authors felt that the F3b differences were fortuitous.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
F2a Offspring - No effect on food consumption of F2a offspring was reported.
F2b Offspring - No effect on food consumption of F2b offspring was reported.

F3a Offspring - No effect on food consumption of F3a offspring was reported.
F3b Offspring - No effect on food consumption of F3b offspring was reported.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
F2a Offspring - no necropsy findings of significance were recorded, but one male pup in he 80-ppm group was found to have had hydrocephalus.
F2b Offspring - no necropsy findings of significance were reported.

F3a Offspring - Gross necropsy did not reveal any remarkable findings.
F3b Offspring - Gross necropsy did not reveal any remarkable findings.
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
F2a Offspring - Except for reduced female fertility in the 750-ppm group (discussed earlier), litter data in all groups were comparable.

F2b Offspring - Except for reduced female fertility in the 750-ppm group (discussed earlier), litter data in all groups were comparable.

F3a Offspring - Litter parameters were not affected by treatment. The study authors concluded that there was nothing to suggest a compound-related effect, although female fertilitywas only 80% and 83% in the DCPD-treated groups, control fertility was worse, only 65%.

F3b Offspring - Litter data in all groups was generally comparable with respect to the various indices. Femalefertility percentages were 85, 80 and 83 for the controls, 80- and 750-ppm groups, respectively. The differences were not considered to be meaningful by the study authors.
Key result
Dose descriptor:
NOAEL
Generation:
F2a
Effect level:
> 80 - < 750 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reproductive Toxicity
Key result
Dose descriptor:
NOAEL
Generation:
F2b
Effect level:
> 80 - < 750 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reproductive Toxicity
Key result
Dose descriptor:
NOAEL
Generation:
other: F3a
Effect level:
750 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reproductive Toxicity
Key result
Dose descriptor:
NOAEL
Generation:
other: F3b
Effect level:
> 80 - < 750 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reproductive Toxicity
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
750 ppm (nominal)
Treatment related:
yes
Relation to other toxic effects:
not specified
Dose response relationship:
yes
Relevant for humans:
not specified

Diet Concentrations

Overall, the  results of the DCPD weekly feed analyse showed  a  69.3 ppm  (87%)  average value for the 80-ppm diet level,  and 693 ppm  (92%)  for the  750-ppm diet level.  Considering the volatility of DCPD,  these results are thought  to indicate reasonable achievement of the intended dietary concentrations.

Table 2. Mean Food Consumption (G) In F1b Rats

Dose (ppm)

Sex

 

Week

4

9

11

20

0

Male

Mean

25

26

26

32

SD

3.8

3.0

3.7

5.7

SE

1.2

1.1

1.4

1.8

N

10

8

7

10

 

80

Male

Mean

26

28

27

30

SD

3.2

1.3

1.7

3.2

SE

1.3

0.46

0.55

1.0

N

6

8

10

10

 

750

Male

Mean

25

25

25

25*

SD

1.9

1.9

0.58

3.2

SE

0.67

0.63

0.20

1.0

N

8

9

8

10

 

0

Female

Mean

22

22

23

29

SD

4.6

4.2

6.0

4.0

SE

1.1

0.89

1.4

1.0

N

17

18

18

16

 

80

Female

Mean

25

25

26

33

SD

2.6

3.6

4.4

6.1

SE

0.91

0.96

1.2

1.4

N

8

14

13

18

 

750

Female

Mean

21

22

21

24*

SD

4.9

4.3

5.0

5.9

SE

1.3

1.0

1.2

1.6

N

15

17

19

14

*p<0.05 as compared to controls: Student's t-test

Table 3. Summary of First Generation – First Mating (F1a)

Indices

Dose (ppm)

0

80

750

Ratio

Percent

Ratio

Percent

Ratio

Percent

Male Fertility

(males producing litter/ mated)

10/10

100

10/10

100

9/10

90

 

Female Fertility

(females producing litter/ mated)

19/20

95

18/20

90

16/20

80

 

Gestation

(females live litter/pregnant)

19/19

100

18/18

100

16/16

100

 

Newborn viability

(live pups/total pups)

209/211

99

210/212

99

198/200

99

 

Pup Viability

(pups Day 4/pups Day 0)

205/209

98

207/210

99

194/198

98

 

Lactation

(pups Day 21/pups Day 4)a

140/140

100

140/140

100

128/128

100

 

Pup Weight in Grams (Mean ± SD)

Day 0 males

7 ± 0.66

7 ± 0.68

7 ± 0.60

Day 0 females

7 ± 0.75

6 ± 0.59

6 ± 0.72

Day 21 males

52 ± 5.4

50 ± 5.6

48 ± 5.1

Day 21 females

49 ± 4.6

46 ± 5.1

46 ± 4.7

Sex Ratio offspring (M/F Day 0)

110/101

111/101

94/106

Live pups per litter (Mean ± SD)

11 ± 3.7

12 ± 2.8

12 ± 2.3

aAfter Litters Reduced

Table 4. Summary of F0 Generation – Second Mating (F1b)

Indices

Dose (ppm)

0

80

750

Ratio

Percent

Ratio

Percent

Ratio

Percent

Male Fertility

(males producing litter/ mated)

10/10

100

10/10

100

10/10

100

 

Female Fertility

(females producing litter/ mated)

18/20

90

18/20

90

19/20

95

 

Gestation

(females live litter/pregnant)

18/18

100

18/18

100

19/19

100

 

Newborn viability

(live pups/total pups)

199/201

99

196/202

97

229/231

99

 

Pup Viability

(pups Day 4/pups Day 0)

193/199

97

187/196

95

216/229

94

 

Lactation

(pups Day 21/pups Day 4)a

128/132

97

128/132

97

139/145

96

 

Pup Weight in Grams (Mean ± SD)

Day 0 males

6 ± 1.1

5 ± 1.6

6 ± 0.61

Day 0 females

6 ± 1.1

5 ± 1.6

6 ± 0.61

Day 21 males

46 ± 7.8

47 ± 8.8

42 ± 6.8

Day 21 females

44 ± 6.9

44 ± 8.2

40 ± 5.7

Sex Ratio offspring (M/F Day 0)

100/101

94/108

108/123

Live pups per litter (Mean ± SD)

11 ± 3.5

11 ± 3.7

12 ± 3.2

aAfter Litters Reduced

Table 5. Summary of F1b Generation – First Mating (F2a)

Indices

Dose (ppm)

0

80

750

Ratio

Percent

Ratio

Percent

Ratio

Percent

Male Fertility

(males producing litter/ mated)

10/10

100

10/10

100

9/10

90

 

Female Fertility

(females producing litter/ mated)

19/20

95

18/20

90

14/20

70

 

Gestation

(females live litter/pregnant)

19/19

100

18/18

100

14/14

100

 

Newborn viability

(live pups/total pups)

241/242

100

209/216

97

162/162

100

 

Pup Viability

(pups Day 4/pups Day 0)

237/241

98

196/209

94

159/162

98

 

Lactation

(pups Day 21/pups Day 4)a

147/150

98

135/139

97

107/109

98

 

Pup Weight in Grams (Mean ± SD)

Day 0 males

6 ± 0.90

6 ± 0.84

6 ± 0.83

Day 0 females

6 ± 0.79

6 ± 0.92

6 ± 0.95

Day 21 males

44 ± 5.9

46 ± 6.4

44 ± 5.5

Day 21 females

41 ± 5.3

43 ± 6.6

42 ± 5.3

Sex Ratio offspring (M/F Day 0)

111/131b

94/122b

84/78b

Live pups per litter (Mean ± SD)

13 ± 2.6

12 ± 2.7

12 ± 2.7

aAfter Litters Reduced

bSome pups mis-sexed

Table 6. Summary of F1b Generation – Second Mating (F2b)

Indices

Dose (ppm)

0

80

750

Ratio

Percent

Ratio

Percent

Ratio

Percent

Male Fertility

(males producing litter/ mated)

10/10

100

10/10

100

9/10

90

 

Female Fertility

(females producing litter/ mated)

19/20

95

19/20

95

17/20

85

 

Gestation

(females live litter/pregnant)

19/19

100

19/19

100

17/17

100

 

Newborn viability

(live pups/total pups)

263/266

99

286/287

100

230/235

98

 

Pup Viability

(pups Day 4/pups Day 0)

250/263

95

280/286

98

214/230

93

 

Lactation

(pups Day 21/pups Day 4)a

149/151

99

149/152

98

127/128

99

 

Pup Weight in Grams (Mean ± SD)

Day 0 males

6 ± 0.84

6 ± 0.63

6 ± 0.54

Day 0 females

6 ± 0.75

6 ± 0.52

6 ± 0.66

Day 21 males

45 ± 6.8

48 ± 7.2

51 ± 6.6

Day 21 females

43 ± 7.4

46 ± 6.6

48 ± 6.6

Sex Ratio offspring (M/F Day 0)

121/145

146/141

119/116

Live pups per litter (Mean ± SD)

14 ± 2.5

15 ± 1.6

14 ± 1.4

aAfter Litters Reduced

Table 7. Summary of F2b Generation – First Mating (F3a)

Indices

Dose (ppm)

0

80

750

Ratio

Percent

Ratio

Percent

Ratio

Percent

Male Fertility

(males producing litter/ mated)

9/10

90

10/10

100

8/9

89

 

Female Fertility

(females producing litter/ mated)

13/20

65

16/20

80

15/18

83

 

Gestation

(females live litter/pregnant)

13/13

100

16/16

100

15/15

100

 

Newborn viability

(live pups/total pups)

162/163

99

195/196

99

204/206

99

 

Pup Viability

(pups Day 4/pups Day 0)

156/162

96

187/195

96

201/204

99

 

Lactation

(pups Day 21/pups Day 4)a

92/100

92

118/118

100

117/120

98

 

Pup Weight in Grams (Mean ± SD)

Day 0 males

6 ± 0.77

6 ± 1.3

7 ± 0.82

Day 0 females

7 ± 0.80

5 ± 1.2

6 ± 0.80

Day 21 males

46 ± 5.8

46 ± 4.7

48 ± 6.1

Day 21 females

45 ± 7.6

42 ± 4.2

45 ± 5.7

Sex Ratio offspring (M/F Day 0)

81/82

103/92

108/98

Live pups per litter (Mean ± SD)

12 ± 3.3

12 ± 3.9

14 ± 2.0

aAfter Litters Reduced

Table 8. Summary of F2b Generation – First Mating (F3b)

Indices

Dose (ppm)

0

80

750

Ratio

Percent

Ratio

Percent

Ratio

Percent

Male Fertility

(males producing litter/ mated)

9/10

90

10/10

100

9/9

100

 

Female Fertility

(females producing litter/ mated)

17/20

85

16/20

80

15/18

83

 

Gestation

(females live litter/pregnant)

17/17

100

16/16

100

15/15

100

 

Newborn viability

(live pups/total pups)

211/215

98

206/213

97

188/191

98

 

Pup Viability

(pups Day 4/pups Day 0)

207/211

98

206/206

100

185/188

98

 

Lactation

(pups Day 21/pups Day 4)a

134/135

99

127/128

99

114/117

97

 

Pup Weight in Grams (Mean ± SD)

Day 0 males

6 ± 0.79

7 ± 0.98

7 ± 0.83

Day 0 females

6 ± 0.64

6 ± 0.87

6 ± 0.83

Day 21 males

49 ± 10

44 ± 11

43 ± 11

Day 21 females

48 ± 9.3

41 ± 12

41 ± 9.5*

Sex Ratio offspring (M/F Day 0)

93/122

107/106

93/98

Live pups per litter (Mean ± SD)

12 ± 2.7

13 ± 2.5

13 ± 2.8

aAfter Litters Reduced

*p<0.05 compared to control: Student’s t-test.

Conclusions:
The authors of the study considered that dietary administration of DCPD at 80 and 750 ppm to three successive generations of rats had no deleterious effects on reproductive performance or the general condition of the animals. However, DCPD was not devoid of reproductive or systemic effects at the 750 ppm dietary level, as previously described.

Thus, the NOAEL in this study is considered to be between 80 - 750 ppm (69 - 693 ppm actual concentration).

Executive summary:

This three generation reproduction non-GLP study in rats using dicyclopentadiene was conducted in 1979, prior to the adoption of the OECD test guidelines for reproductive and developmental toxicity. However the methods used and overall quality of the study were considered similar to OECD 416 and the study was considered adequate for assessment.

 

F0 rats were mated seven weeks after initiation of treated diet. Selected F1b pups were designated F1 parents and were approx. 100 days old when mated to produce the F2a litters and subsequently the F2b litters. Selected F2b pups were designated F2 parents and similarly used to produce the F3 a and b litters. Animals were exposed to nominal dietary concentrations of 0, 80, 750 ppm DCPD.

 

The authors of the study considered that dietary administration of DCPD at nominal concentrations of 80 and 750 ppm to three successive generations of male and female albino rats had no deleterious effects on reproductive performance or the general condition of the animals, in comparison to performance of control rats maintained concurrently. However, DCPD was not devoid of reproductive or systemic effects at the 750 ppm dietary level. Mean food consumption at 20 weeks in the F1B parents was reduced in both sexes in a treatment-related manner, with statistical significance at the 750 ppm level. At 750 ppm, female fertility was reduced in the F2A and F2B generations, however, the differences from control were not statistically significant, and this may have been due to one male in the 750 ppm group that failed to sire litters in either mating. A treatment- related reduction in mean pup weight on PND 21 was noted in the F3B generation, with mean m/f pup weights of 49/48, 44/41, and 43/41* grams in the control, 80 and 750 ppm groups, respectively.

 

Thus, the NOAEL in this study is considered to be between 80 - 750 ppm (69 - 693 ppm actual concentration).

Endpoint:
one-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
supporting 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 published report (in Japanese with abridged English translation) and summary information sheet (provided by Japan Chemical Industry Ecology-Toxicology and Information Center (JETOC)), adequate for assessment.
Reason / purpose:
reference to same study
Qualifier:
according 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 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
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: up to 7 days
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
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
Details on study schedule:
- 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.

- The test substance was administered to male and female rats daily by oral gavage from 2 weeks prior to mating and during mating (approx. 2 weeks).
- Male rats continue to be dosed until sacrifice of females after day 3 of lactation. Females continue to be dosed through gestation to day 3 of lactation.
- Females were sacrificed on day 4 of lactation and males on day 45 of the study.
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
4 mg/kg bw/day
Dose / conc.:
20 mg/kg bw/day
Dose / conc.:
100 mg/kg bw/day
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.
Parental animals: Observations and examinations:
CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: Yes

FOOD EFFICIENCY: No

WATER CONSUMPTION: 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: glutamic oxaloacetic transaminase (GOT), glutamic-pyruvate transaminase (GPT), alkaline phosphatase (ALP), gamma-glutamyl transpeptidase (¿-GTP), urea nitrogen, glucose, total cholesterol, triglycerides, creatinine, total bilirubin, total protein, albumin, A/G ratio, calcium, inorganic phosphorus, sodium, potassium, chloride

PREGNANCY DATA: number of pairs with successful mating, mating index (%), number of pregnant females, fertility index (%), pairing days until mating, number of females with live pups, gestation index (%), gestation length, number of corpora lutea, number of implantation sites, implantation index (%), delivery index (%)
Oestrous cyclicity (parental animals):
yes
Sperm parameters (parental animals):
No
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in offspring: number and sex of pups, stillbirths, live pups on day 0, live birth index (%), number of live pups on day 4, viability index on day 4 (%), bodyweight of pups on days 0 and 4, bodyweight gain days 0-4

GROSS EXAMINATION OF PUPS: Yes (on day 4)
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: all surviving animals on day 45
- Maternal animals: day 4 of lactation

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
Postmortem examinations (offspring):
Gross examination on day 4
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. Fisher's exact method for Mating rate, fertility rate, birth rate, sex ratio (male / female)
Reproductive indices:
mating index, fertility index, gestation index, implantation index
Offspring viability indices:
delivery index, live birth index, viability index (day 4)
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
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.
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
DCPD had no effects on reproductive parameters such as: mating index, fertility index, gestation length, number of corpora lutea or implantations, implantation index, gestation index, delivery index or parturition. However, two dams in the 100 mg/kg group had total litter loss during the lactation period.
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- 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 FOOD CONSUMPTION (PARENTAL ANIMALS)
Males and surviving females showed slight suppression of body weight gain and decreased food consumption.

ORGAN WEIGHTS (PARENTAL ANIMALS)
- There were increased liver and kidney weights in male rats given 100 mg/kg.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- Two females in the 100 mg/kg group lost 100% of their litters during lactation (days 1-4).

HISTOPATHOLOGY (PARENTAL ANIMALS)
- 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 group. Similar histopathological changes were seen in kidneys of 4, 20 mg/kg group male rats and in adrenals of 20 mg/kg group male rats.

OTHER FINDINGS (PARENTAL ANIMALS)
- Blood chemistry of high dose males showed increase in GOT and GPT; no test material related changes occurred in haematology parameters for any treatment group.
Dose descriptor:
NOAEL
Effect level:
4 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Systemic Toxicity
Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Systemic and Reproductive Toxicity
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Reproductive Toxicity
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
Clinical signs:
no effects observed
Description (incidence and severity):
No abnormal findings ascribable to the compound were found for external features, clinical signs, or on necropsy of the offspring.
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
Examination of neonates revealed a low viability index for the 100 mg/Kg bw/dose group.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Lower birth weight and lower body weights were observed in the 100 mg/Kg bw/day dose group.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No abnormal findings ascribable to the compound were found for external features, clinical signs, or on necropsy of the offspring.
Histopathological findings:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
A low viability index and tendency to lower birth weight and body weight gain was observed in neonates in the highest dose group (100 mg/kg), a dose level that was associated with reduced food consumption, reduced weight gain, and mortality (2/10) in females. No significant differences in number of offspring, live offspring at birth, sex ratio or live birth index were found. However, two dams in the 100 mg/kg group had total litter loss during the lactation period. No abnormal findings were observed in external features, clinical signs in offspring, or at necropsy of offspring.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
20 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reproductive and Developmental toxicity
Critical effects observed:
no
Reproductive effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day
Treatment related:
yes
Relation to other toxic effects:
not specified
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.

 

Table 6. Fertility and Pregnancy Data in Rats

Dose (mg/Kg/day)

0

4

20

100

Number of animals

10

10

10

8

Number of Pairs Examined

10

10

10

8

Number of Pairs with Successful mating

10

10

9

8

Mating Index (%)a

100

100

90

100

Number of Pregnant Females

9

9

5

7

Fertility Index (%)b

90

90

55.6

87.5

Pairing days until Mating

2.2 ± 1.03c

2.2 ± 1.14

1.8 ± 0.83

3.0 ± 1.07

Number of Estrous Stages without Mating

0.0 ± 0.00

0.0 ± 0.00

0.0 ± 0.00

0.0 ± 0.00

a Mating Index (%) = (Number of pair with successful mating / Number of pairs examined) x 100

b Fertility Index (%) = (Number of pregnant animals / Number of pairs with successful mating ) x 100

c Values are expressed as Means ± S.D.

 

Table 7. Delivery and Litter Data

Dose (mg/Kg/day)

0

4

20

100

Number of females examined

9

9g

5

7

Number of females with live pups

8

8

5

7

Gestation Index (%)a

100

88.9

100

100

Gestation Length (days)

22.3 ± 0.50f

22.6 ± 0.52

22.2 ± 0.45

22.1 ± 0.38

Number of Corpora Lutea

18.0 ± 2.06

15.7 ± 4.80

18.8 ± 3.27

17.9 ± 2.55

Number of Implantation Sites

17.8 ± 1.72

13.6 ± 6.50

14.4 ± 4.28

15.9 ± 4.02

Implantation Index (%)b

98.9

79.7

77.6

87.6

Delivery Index (%)c

93.4

81.2

92.6

94.0

Number of Pups Delivered

16.7 ± 2.55

14.0 ± 4.57

13.2 ± 3.63

15.0 ± 4.00

Number of Live Pups on Day 0

16.6 ± 2.55

13.5 ± 4.38

13.2 ± 3.63

14.1 ± 3.98

Live Birth Index (%)d

99.3

97.0

100

94.7

Sex Ratio (male/female)

0.97 (74/76)

1.27 (62/49)

0.69 (27/39)

0.84 (48/57)

Number of Live Pups on Day 4

16.2 ± 2.29

13.4 ± 4.31

13.2 ± 3.63

9.1 ± 7.01*

Viability Index on Day 4 (%)e

98.9

99.2

100

66.1**

Body Weight of Pups (g)

On Day 0

 

Males

6.4 ± 0.72

6.9 ± 0.70

6.6 ± 0.51

5.6 ± 0.81

Females

6.1 ± 0.75

6.8 ± 0.70

6.1 ± 0.34

5.4 ± 0.79

On Day 4

 

Males

10.1 ± 1.25

10.8 ± 1.25

10.7 ± 1.86

8.8 ± 1.92

Females

9.7 ± 1.30

10.7 ± 1.25

9.9 ± 1.44

8.4 ± 1.95

Body Weight Gain of Pups (g)

Day 0 to 4

 

Males

3.6 ± 0.64

4.0 ± 0.58

4.1 ± 1.54

2.9 ± 1.39

Females

3.6 ± 0.64

4.1 ± 0.64

3.8 ± 1.12

2.8 ± 1.31

a Gestation Index (%) = (Number of females with live pups / Number of pregnant females) x 100

b Implantation Index (%) = (Number of implantation sites / Number of corpora lutea) x 100

c Delivery Index (%) = (Number of pups delivered / Number of implantation sites) x 100

d Live Birth Index (%) = (Number of live pups on Day 0 / Number of pups delivered) x 100

e Viability Index (%) = (Number of live pups on Day 4 / Number of live pups on Day 0) x 100

f Values are expressed as Mean ± S.D.

g One female was not examined because the pregnancy was noted at the time of necropsy after the mating period.

Conclusions:
The NOAEL for reproductive/developmental effects in the F1 generation pups and parental females was 20 mg/Kg/day. The NOAEL for reproductive/developmental effects in the parental males was 100 mg/Kg/day.
Executive summary:

The study was conducted in accordance with OECD guideline 422 and complies with GLP requirements. It is available as a study report (in Japanese with an abridged English translation). A detailed summary of the study report was also published by Japan Chemical Industry Ecology-Toxicology and Information Center (JETOC).

DCPD was administered by gavage at 0 (olive oil vehicle), 4, 20, or 100 mg/Kg/day to groups of 10 male and 10 female SD rats (Crj: CD). DCPD was dissolved in olive oil and administered by gavage using a stomach tube once per day in the morning at a dose volume of 5 mL/Kg adjusted for most recent body weight measurement. Males were dosed for 44 days while females were dosed from 14 days before mating until day 3 of lactation. Males were sacrificed on day 45 while females were killed on lactation day 4. After 14 days of exposure males were co-housed with one female for 7 days or until mating was confirmed by copulatory plug.

 

Dicyclopentadiene induced signs of systemic toxicity (suppression of body weight gain and decreased food consumption) in male and female rats at the 100 mg/Kg/day dose level. No compound-related effects were seen on reproductive parameters such as mating index, fertility index, gestation length, number of corpora lutea or implantations, implantation index, gestation index, delivery index or parturition. However, two dams in the 100 mg/Kg group had total litter loss during the lactation period. Effects on neonates included low viability index, lower birth weight and body weight gain in the 100 mg/Kg group, but not at lower dose levels.

 

The NOAEL for reproductive/developmental effects in the F1 generation pups and parental females was 20 mg/Kg/day. The NOAEL for reproductive/developmental effects in the parental males was 100 mg/Kg/day.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
Study duration:
subchronic
Species:
rat
Quality of whole database:
Three generation reproductive toxicity study (Klimisch score = 2). The NOAEL was considered to be between 80 - 750 ppm (69 - 693 ppm actual concentration).
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Non-human information

In a key three generation reproduction study (Litton Bionetics, LBI Project no. 10734 -07, 1979, pre-GLP) with two litters per generation, dicyclopentadiene (DCPD) was administered to Sprague Dawley rats (10 males and 20 females per dose group) in the diet at nominal concentrations of 0, 80 or 750 ppm. DCPD was suspended in 300 ml corn oil and blended with 10 kg of the basal diet. Samples of each diet were analysed at weekly intervals by gas-liquid chromatography. The results showed actual average dietary concentrations of 69.3 ppm (87% of nominal) and 693 ppm (92% of nominal) for the low and high dietary groups. The second and third generations were derived from the second (b) litters of the previous generation. 

One F0 female from the 80 ppm group was found dead on study week 28 but all other F0 rats survived until the end of the study. Mean food consumption at 20 weeks in the F1B parents was reduced in both sexes in a treatment-related manner, with statistical significance at the 750 ppm level. At 750 ppm female fertility was reduced in the F2A and F2B generations, however, the differences from control were not statistically significant and this may have been due to one male in the 750 ppm group that failed to sire litters in either mating. A treatment-related reduction in mean pup weight on PND 21 was noted in the F3B generation with mean m/f pup weights of 49/48, 44/41, and 43/41* grams in the control, 80 and 750 ppm groups, respectively.

The authors of the study considered that dietary administration of DCPD at 80 and 750 ppm to three successive generations of rats had no deleterious effects on reproductive performance or the general condition of the animals. However, DCPD was not devoid of reproductive or systemic effects at the 750 ppm dietary level, as described above. Thus, the NOAEL is considered to be between 80 - 750 ppm (69 - 693 ppm actual concentration).

According to the BG Chemie Toxicological Evaluation No. 84 (dicyclopentadiene), the highest concentration (693 ppm) was judged equivalent to a daily intake of 50 mg/kg body weight at an assumed feed intake of 70 g/kg body weight/day. Consideration of the reported food and body weight data at week 4 (when the amount of dicyclopentadiene consumed would be high in relation to body weight) indicates that males of 300g body weight consuming 25g diet would receive 58 mg/kg/ body weight/day and females of 200g body weight consuming 25g diet would receive 87 mg/kg/ body weight/day. The value for females would not be exceeded during pregnancy.

In a supporting OECD test guideline 422 (combined repeated dose toxicity study with the reproduction/developmental toxicity screening test), dicyclopentadiene (DCPD), was administered to rats by oral gavage at dose levels of 0, 4, 20 or 100 mg/kg/day. This study was conducted in compliance with GLP at the Mitsubishi Chemical Safety Inst., Kashima Laboratory, Japan. The doses used were selected following a dose range-finding study, in which male and female rats were orally dosed for 10 days with 0, 30, 100 or 300 mg/kg/d. The high dose in the range-finding study produced lethality, so the top dose for the OECD 422 study was set at 100 mg/kg/d. Although the study report is written in Japanese, the study details were summarized in English by the Japan Chemical Industry Ecology-Toxicology and Information Centre JETOC (1998a), and an abridged English translation of the GLP study report is also available.

The study design for OECD Guideline 422 includes administration of the test substance for two weeks prior to the mating of male and female rats and, during mating (approximately 2 weeks). The females continue to be dosed throughout gestation and until termination on day 4 of lactation. The males are dosed through to approximately day 45 of the study.

In the 100 mg/kg/d group, two females died. In these decedents the following findings were observed: lung congestion, enlargement of the adrenal gland, bleeding in the thymus and bleeding in the gastric mucosa. In addition, signs of systemic toxicity were seen in both males and females in the 100 mg/kg/day group, including transient salivation, depressed body weight gain and decreased food consumption. An increase in the incidence of fatty droplets in the fascicular zone of the adrenals in males and females in the high dose group and in males in the mid-dose group were reported to be of unknown toxicological significance. Based on these observations, the NOAEL for systemic toxicity of the parental animals in this study appears to have been 4 mg/kg/d for males and 20 mg/kg/d for females.

DCPD had no effects on reproductive parameters such as: mating index, fertility index, gestation length, number of corpora lutea or implantations, implantation index, gestation index, delivery index or parturition. However, two dams in the 100 mg/kg/d group had total litter loss during the lactation period. Examination of the neonates in the 100 mg/kg/d group revealed a low viability index, lower birth weight and reduced body weight gain. The number of live pups on PND 4 and the viability index were: 16.3/98.9%; 13.4/99.2%; 13.2/100% and 9.1*/66.1**% in the 0, 4, 20 and 100 mg/kg/d groups, respectively. There were no significant differences in the number of offspring or live offspring at birth, the sex ratio or the live birth index. Exposure to DCPD produced no effects on external features, clinical signs or necropsy of the offspring. Based on these results, the NOAEL for parental females and offspring appears to be 20 mg/kg/d and for parental males is 100 mg/kg/d.

The reproductive effects of DCPD were also evaluated in a continuous breeding study in Sprague-Dawley rats performed by US NTP (Jamieson et al., 1995). Although a Society of Toxicology abstract is available for this study, the full study report is not available, and therefore it is considered to be Klimisch category 4. In this study oral (gavage) doses of 100 mg/kg/d DCPD was shown to produce reproductive effects such as reduced pup body weight, increased pup mortality and decreased pup survival in F1 litters. Effects in the F2 generation were similar to those in F1. It is uncertain if the F1 -F2 pup body weight reductions (9 -12%) were secondary to maternal toxicity.

Effects on developmental toxicity

Description of key information

There are no human data available for developmental effects of dicyclopentadiene in humans. A key developmental toxicity study using the dietary rout of exposure to dicyclopentadiene is available together with data from dose range finding studies in pregnant rats and rabbits using the oral (gavage) route of exposure. These latter studies do not include foetal skeletal or visceral examinations.

 

There are no developmental toxicity studies available using the dermal or inhalation routes of exposure to dicyclopentadiene. 

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1977-06-13 to 1978-06-06
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Pre-GLP (1978), animal experimental study, minor deviations in design and/or reporting but otherwise adequate for assessment.
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
EPA OPP 83-3 (Prenatal Developmental Toxicity Study)
GLP compliance:
no
Remarks:
pre-GLP (1978)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: MC/B (2909 Highland Avenue, Norwood, Ohio 45212); Catalog No. TX 310
- Expiration date of the lot/batch: Not specified
- Purity test date: Analysis of DCPD was performed with a UC-W98 column post receipt on August 18, 1976. (Purity: 98 to 99%)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Closed container in the freezer
- Stability under test conditions: Stability analysis revealed that in the closed containers, the concentration dropped 27.6% for the 80 ppm level and 30.8% for the 750 ppm level over a 10-day period.
- Solubility and stability of the test substance in the solvent/vehicle: Because of poor water solubility, DCPD was prepared for administration to animals by dissolving it in corn oil (Mazola).

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: waxy solid converted to an easily measured liqud by slight warming

FORM AS APPLIED IN THE TEST (if different from that of starting material): waxy solid converted to an easily measured liquid by slight warming

Species:
rat
Strain:
Sprague-Dawley
Remarks:
CRL:COBS CD (SD) BR
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: CRL:COBS(SD)BR
- Source: Charles River Breeding Laboratories, Inc., Portage, Michigan, USA
- Age at start of treatment: 11 weeks
- Housing: Individually housed in wire cages
- Diet: Purina Laboratory Chow ad libitum
- Water: acidified pH 2.5 ad libitum
- Acclimation period: 12 days prior to pairing for mating

ENVIRONMENTAL CONDITIONS
- Temperature controlled: no data
- Humidity: no data
- Air changes (per hr): no data
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: Not specified
Route of administration:
oral: feed
Vehicle:
corn oil
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): The test material was incorporated into the basal-diet (Purina Laboratory Chow) on gestation Days 6 through 15 so as to provide the appropriate dose levels.
- Mixing appropriate amounts with (Type of food): The test material (0.8, 2.5 or 7.5 g) was suspended in 300 mL of corn oil and blended with 10 Kg of the basal diet in a twin shell blender for 15 minutes. The control diet contained 300 mL of corn oil per 10 Kg of meal.
- Storage temperature of food: Not specified

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil; Because of poor water solubility, DCPD was prepared for administration to animals by dissolving it in corn oil (Mazola).
- Concentration in vehicle: The test material (0.8, 2.5 or 7.5 g) was suspended in 300 mL of corn oil.
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
- M/F ratio per cage: 1:1
- Females were acclimated to laboratory conditions for 12 days and then paired with a sexually mature male of the same strain and from the same supplier
- Proof of pregnancy: Females were examined daily for the presence of a copulatory plug as evidence of mating, designated Day 0 of gestation
Duration of treatment / exposure:
Days 6-15 of gestation
Frequency of treatment:
Daily
Duration of test:
Days 0-19 of gestation. Although the protocol for the study indicated that the female rats were to be sacrificed on gestation day 20, they were inadvertently killed on day 19. This deviation was not considered to affect the integrity of the study.
Dose / conc.:
0 ppm
Remarks:
Group 1 (Control)
Dose / conc.:
80 ppm
Remarks:
Group 2
Dose / conc.:
250 ppm
Remarks:
Group 3
Dose / conc.:
750 ppm
Remarks:
Group 4
No. of animals per sex per dose:
20 females/concentration
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: A clear rationale for selection of the doses that were used in the study was not provided in the study report. However, the study report does mention that 'The
dose levels used in this study were approved by Dr. E. Ross Hart of LBI based on previous studies'.
- Rationale for animal assignment (if not random): Not specified. The report only mentions that 'Mated female rats were assigned sequentially to treatment groups and identified by cage cards'
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The mated female rats were observed daily for changes in general appearance, behaviour and condition

BODY WEIGHT: Yes
- Time schedule for examinations: The mated female rats were weighed on Days 0, 6, 16 and 19 of gestation

FOOD CONSUMPTION: Yes
- Food consumption was measured during the period 0-6, 6-16 and 16-19 days of gestation

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

POST-MORTEM EXAMINATIONS: Yes
- On Day 19 of gestation the female rats were necropsied
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
- The number of implantation sites and their placement in the uterine horns, live and dead foetuses and resorption sites were recorded

Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: No data
- Number of implantations: Yes
- Number of resorptions: Yes
Fetal examinations:
- External examinations: Yes: The foetuses were removed, examined externally for abnormalities and weighed.
- Soft tissue examinations: Yes: One third of the foetuses of each litter were fixed in Bouin's fluid. These were later examined for changes in the soft tissues of the head, thoracic and visceral organs.
- Skeletal examinations: Yes: The remaining foetuses of each litter were examined for skeletal abnormalities following staining with Alizarin Red S.
- Head examinations: No data
Statistics:
Statistical analysis of the data was performed using the litter as a basic sampling unit. Dunnett's t-test was used to determine statistical significance (p<0.05) with regard to difference between means with near normal distribution (maternal body weights and food consumption, mean pup weight based on litter averages). Ratios, e.g. sex ratio and pregnancy ratio, were analysed with a 2x2 contingency table with Yates' correction. With regard to discontinuous parameters as measured by the number of abnormal foetuses within a litter, Wilcoxon Rank Sum was used.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
One low dose dam was emaciated and had an arched back and a red crust around the nose and mouth on Day 19 of gestation. All other control and test group dams were normal in appearance throughout the study.
Mortality:
no mortality observed
Description (incidence):
There were no deaths reported through the study period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean body weight and food consumption showed no significant differences between control and treated rats. On gestation day 19, body weights of dams in the control, 80, 250 and 750 ppm groups were 350, 334, 346, 342 grams, with body weight gain from GD 0-19 of 32, 20, 23, 29 grams, respectively.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Mean body weight and food consumption showed no significant differences between control and treated rats. On gestation day 19, body weights of dams in the control, 80, 250 and 750 ppm groups were 350, 334, 346, 342 grams, with body weight gain from GD 0-19 of 32, 20, 23, 29 grams, respectively.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Examination of the animals at necropsy revealed a dark red area in the lungs of rat no. 6650 (Group 2) and a liver mottled with small white spots in animal no. 6698 (Group 4). These changes were not considered to be treatment-related.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The number of implantation sites (Left horn/Right horn) were 154/159; 132/168; 132/160; & 134/158 in the control, 80 ppm, 250 ppm, and 750 dose groups, respectively.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
The number of litters with resoprtions were 14 (74%), 8 (40%), 11 (58%), & 8 (42%) in the control, 80 ppm, 250 ppm, and 750 dose groups, respectively.
Early or late resorptions:
no effects observed
Description (incidence and severity):
The number of resorptions were 18, 22, 19, & 13 in the control, 80 ppm, 250 ppm, and 750 dose groups, respectively.
Dead fetuses:
no effects observed
Description (incidence and severity):
There were no dead fetuses at any of the concentrations tested.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
The pregnancy ratio (Pregnant/Bred) was 19/21, 20/20, 19/20, & 19/20 in the control, 80 ppm, 250 ppm, and 750 dose groups, respectively.
Details on maternal toxic effects:
Maternal toxic effects:no effects
Key result
Dose descriptor:
NOAEL
Effect level:
750 ppm (nominal)
Based on:
test mat.
Basis for effect level:
other: Systemic Toxicity
Key result
Dose descriptor:
NOAEL
Effect level:
750 ppm (nominal)
Based on:
test mat.
Basis for effect level:
other: Reproductive Toxicity
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Average fetal weight (in grams) was 2.3, 2.3, 2.4, & 2.4 in the control, 80 ppm, 250 ppm, and 750 dose groups, respectively.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
No mortality was observed through the study period and the percentage of live fetuses (per implantatio site) was 295/313 (94%); 278/300 (93%); 273/292 (93%); & 279/292 (96%) in the control, 80 ppm, 250 ppm, and 750 dose groups, respectively.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex ratio did not differ significantly between treated and control groups.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
The mean live litter size was 15.5, 13.9, 14.4, & 14.7 in the control, 80 ppm, 250 ppm, and 750 dose groups, respectively.
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Examination of the offspring at delivery revealed subcutaneous hematomas in fetuses from litters at all dose levels (See Table 4). Although there was a significant decrease in the number of fetuses with subcutaneous hematomas in Group 2 (80 ppm) this was not judged to be a dose-related response by the study authors.

Observations on fetuses at delivery included one fetus of female no. 6622 (Group 1) with swelling of the right hind limb and one fetus of female no. 6617 (Group 1) with intestines protruding at the umbilicus.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The study authors concluded that while most of the changes noted during the skeletal examination (See Table 5) were not strictly normal, have been frequently observed in 19 day old rat fetuses of this strain and source in the laboratory.

The study report states that the unusual changes for the most part were related to retarded bone ossification and were not malformations as such. Neither the frequency nor the character of these changes indicated an adverse effect on foetal growth and development, or a teratogenic potential.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Examination of the Bouin's fixed specimens revealed (in addition to the previously mentioned protruding intestines in the fetus of litter no. 6617) the absence of the left kidney in one fetus of litter no. 6620 (Group 1), enlarged kidneys in one fetus of litter no. 6645 (Group 2), and unilateral anophthalmia in one fetus of litter no. 6709 (Group 4). These changes were not considered to be indicative of a dose-related response.
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects
Key result
Dose descriptor:
NOAEL
Effect level:
750 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Developmental Toxicity
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

750 ppm in the diet is equivalent to 60 mg/Kg bw/day based on a 250 g rat consuming 20 g diet/day.

Based on the final body weight and food consumption data, the dose in mg/Kg in the 750 ppm group is as follows: 

26 g/day of diet consumed;

Diet was prepared with 7.5 g DCPD/10 Kg feed = 0.75 g/Kg = 750 mg/Kg = 0.75 mg/g;

0.75 mg/g X 26 g/d = 19.5 mg/d;                          

19.5 mg/0.342 Kg (d19 high dose mean bw) = approx. 60 mg/Kg

Table 2. Body Weights and Food Consumption of Pregnant Rats

Dose (ppm)

 

Mean Body Weights in Gramsa

Mean Daily Food Consumption

in Gramsa

 

Day 0

Day 6

Day 15

Day 19

Day 0-6

Day 6-15

Day 16-19

0

Mean

218

244

296

350

19

20

26

SD

13

13

18

20

4

4

3

SE

3.1

3.0

4.1

4.7

1

1.1

0.8

N

19

19

19

19

17

11

19

 

80

Mean

214

244

289

334

20

19

23

SD

18

13

34

45

4

3

5

SE

4.1

2.9

7.5

10.0

1.0

0.8

1.2

N

20

20

20

20

15

18

20

 

250

Mean

223

247

295

346

22

20

26

SD

12

18

15

17

5

1

4

SE

2.8

4.2

3.4

3.8

1.2

0.3

0.9

N

19

19

19

19

15

11

18

 

750

Mean

213

243

291

342

22

20

26

SD

18

14

19

27

4

1

3

SE

4.1

3.1

4.1

6.3

1.0

0.3

0.6

N

19

19

19

19

16

11

19

aCalculations do not include non-pregnant females

Table 3. Summary of Reproductive Performance

 

Dose (ppm)

0

80

250

750

Pregnancy Ratio

(Pregnant/Bred)

19/21

20/20

19/20

19/20

Live Littersa

19/19

(100%)

20/20

(100%)

19/19

(100%)

19/19

(100%)

Implantation Sites (Left Horn/Right Horn)a

154/159

132/168

132/160

134/158

Resorptionsb

18

22

19

13

Litters with Resorptionsa

14 (74%)

8 (40%)

11 (58%)

8 (42%)

Dead Fetusesb

0

0

0

0

Litters with Dead Fetusesa

0

0

0

0

Live Fetuses / Implantation Sitea

295/313

(94%)

278/300

(93%)

273/292

(93%)

279/292

(96%)

Mean Live Litter Size (Fetuses)c

15.5

13.9

14.4

14.7

Average Fetal Weight (g)c

2.3

2.3

2.4

2.4

Average Fetal Length (cm)c

2.7

2.6

2.7

2.7

a analyzed with a 2 x 2 contingency table with Yates' correction

b analyzed using Wilcoxon Rank Sum test

c Dunnett's t-test

Table 4. Incidence of Subcutaneous Hematomas

Dose (ppm)

Number of Fetuses with Subcutaneous Hematomas (litters)

0

41 (10)

80

15 (7)

250

29 (11)

750

34 (14)

 

Table 5. Results of the Skeletal Examination

Dose (ppm)

Number of Fetuses Examined

Number of Fetuses Normal

Fetuses with commonly encountered changes only

Fetuses with unusual skeletal variations

0

199a(19)b

106

91 (17)

2 (2)

80

192 (20)

85

103 (19)

4 (3)

250

187 (19)

92

95 (13)

0 (0)

750

192 (19)

91

98 (17)

3 (2)

aTwo specimens of litter no. 6634 lost during processing and handling, not examined

bNumber of litters in parentheses

Conclusions:
The NOAEL for maternal and developmental toxicity was 750 ppm.
Executive summary:

This teratology study in the rat was conducted prior to the publication of OECD test guideline 414 and the adoption of GLP requirements. However, in most respects, the methods used are similar to OECD 414 and the study is considered to be Klimisch Cat 2 and is considered adequate for the assessment of the developmental toxicity of DCPD.

Groups of 20 pregnant female rats each were fed diets containing nominal DCPD concentrations of 0, 80, 250, 750 ppm from gestation day 6 to 15. The 750 ppm dietary concentration is approximately 60 mg/Kg/day, based on final body weight and food consumption.

Dietary administration of DCPD at 80, 250 and 750 ppm produced no effects on the pregnant dams. There was no evidence of teratogenicity or developmental toxicity at dietary doses of up to 750 ppm (approx. 60 mg/Kg bw/day).

Thus, the NOAEL for both maternal and developmental toxicity is considered to be 750 ppm.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
60 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Neither maternal toxicity nor developmental toxicity was observed at any dietary level in a key developmental toxicity study with Sprague Dawley rats. Therefore,750 ppm was considered to be the NOAEL. Consideration of the reported food and body weight data for days 6-15 of gestation indicates that females with a body weight of 250 g consuming 20 g diet/day would receive 60 mg DCPD/Kg body weight/day. On that basis, the oral (dietary) NOAEL for maternal and developmental toxicity is considered to be 60 mg/Kg/day.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Non-human information

A developmental toxicity study with dicyclopentadiene (DCPD) in Sprague-Dawley rats was conducted using the dietary route of oral exposure (Litton Bionetics, 1978). This is considered to be the key study and includes standard (guideline) methods of evaluation. Dietary concentrations of 0, 80, 250 or 750 ppm were fed to groups of 20 time-mated rats on days 6-15 of gestation (where day 0 was the day of confirmation of mating). There was no maternal toxicity and no developmental toxicity at any dietary level and therefore 750 ppm was a NOAEL.

According to the BG Chemie Toxicological Evaluation No. 84 (Dicyclopentadiene), the highest concentration (750 ppm) was judged equivalent to a daily intake of 150 mg/kg body weight at an assumed feed intake of 200 g/Kg body weight/day. Consideration of the reported food and body weight data for days 6-15 of gestation indicates that females of 250 g body weight consuming 20 g diet would receive 60 mg DCPD/Kg body weight/day. On the basis of this study, the oral (dietary) NOAEL for maternal and developmental toxicity is 60 mg/Kg/day.

Limited data are available for the oral (gavage) route of administration of dicyclopentadiene. Dose range finding studies have been conducted in pregnant rats and rabbits (Gulati, 1993). These provide data on maternal toxicity but little information on developmental toxicity because there was no visceral or skeletal examination of the foetuses. Groups of 11 time-mated rats were dosed with 0, 50, 200, 300, 400 or 500 mg/Kg/day on days 6-15 of gestation (where day 0 was the day of confirmation of mating). Dose levels of 300 mg/Kg/day or more caused maternal lethality; a single death occurred in the 200 mg/kg/day group. Reduced body weight gain was observed in females given 50 or 200 mg/Kg/day. Lower foetal weight was associated with 200 mg/Kg/day but not with 50 mg/Kg/day. A NOAEL for maternal toxicity was not established in this study.

 

Groups of 10 mated New Zealand White rabbits were dosed with 0, 25, 100, 200, 300 or 400 mg/Kg/day on days 6-19 of gestation (where day 0 was the day of mating). Signs of systemic toxicity and lethality were observed in the 300 and 400 mg/Kg/day groups. Maternal body weight gain was reduced in the 200 mg/Kg/day group. The abortion of one litter in the 100 mg/Kg/d group occurred in the absence of a statistically-significant reduction in maternal body weight, and no data for food consumption is provided in this DRF study. Consequently, it is uncertain if the abortion seen in one dam at 100 mg/Kg was due to a direct effect of DCPD on the foetuses in this litter, or as the consequence of maternal toxicity at 100 mg/Kg.

Justification for classification or non-classification

Several of the developmental and reproductive toxicity studies with dicyclopentadiene (DCPD) suggest that it produced foetotoxic or developmental effects at doses at or below those that produced clear signs of maternal toxicity, as summarised below:

Litton Bionetics Rat 3 Generation Reproductive Toxicity, LBI PROJECT No. 10734-07 

Treatment-related reduction in mean pup weight at weaning was noted in the F3B generation, with mean M/F pup weights on PND 21 of 49/48, 44/41, and 43/41* grams for the control, 80 and 750 ppm groups, respectively. The study authors stated these effects as “fortuitous” since mean pup weights in the other generations were not appreciably different from controls. Female pup weight on PND 21 was statistically significantly lower than control.

 

OECD 422 Screening 28 day and Reproductive Rat Toxicity Study, Mitsubishi Chemical Safety Institute Ltd., Kashima Laboratory

Two dams in the 100 mg/kg group had total litter loss during the lactation period. Examination of the neonates in the 100 mg/Kg group revealed a low viability index, lower birth weight and reduced body weight gain. The number of live pups on PND 4 and the viability index were: 16.3/98.9%, 13.4/99.2%, 13.2/100%, and 9.1*/66.1**% in the 0, 4, 20 and 100 mg/Kg/day groups, respectively. Based on the observations in the study the NOAEL for parental females and offspring appears to be 20 mg/Kg/day and for parental males is 100 mg/Kg/day.

Jamieson et al. (1995) Rat Continuous Breeding Study (SOT poster abstract)

DCPD was shown to produce effects such as reduced pup body weights, increased pup mortality and decreased pup survival in F1 litters at 100 mg/Kg. Effects seen in F2 were not greater than in F1. The abstract contains no specific mention of systemic toxicity in the adult females. Consequently, it is uncertain whether the 9% and 12% pup weight reductions in F1 and F2 pups (respectively) from DCPD-treated females was secondary to maternal toxicity. Since a full report is not available the study quality cannot be assessed.

 

Gulati, D.K. et al. (1993). Range-finding studies: Developmental toxicity of dicyclopentadiene when administered via gavage to New Zealand White rabbits. Study No. NTP-92-RF/DT-044. 

The abortion of one litter in the 100 mg/Kg/d group occurred in the absence of a statistically-significant reduction in maternal body weight, and no data for food consumption is provided in this DRF study. Consequently, it is uncertain if the abortion seen in one dam at 100 mg/Kg was due to a direct effect of DCPD on the foetuses in this litter, or as the consequence of maternal toxicity at 100 mg/Kg.

 

Section 3.7 of the ECHA Guidance on the Application of the CLP Criteria Version 4.1, June 2015 (https://echa.europa.eu/documents/10162/13562/clp_en.pdf) provides information related to Category 2 reproductive toxicity and the assessment of maternal toxicity. Based on the overall weight of evidence, it is proposed to classify DCPD as a Category 2

reproductive toxicant, based upon foetotoxic effects such as reduced pup body weight, increased pup mortality, and decreased pup survival observed at oral doses equal to or below those that produced significant maternal toxicity in adult females.