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Toxicity to reproduction

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Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Remarks:
GLP test according to guideline. The test report from the Japanese Ministry of Health & Welfare is not available, but all the data were validated by the Japanese authorities within the OECD SIDS program Information in this record is from therefore from the SIDS dossier, a reliable secondary source, and used as supportive information. Reliability assessment based on that given in SIDS dossier.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1999

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
5-ethylidene-8,9,10-trinorborn-2-ene
EC Number:
240-347-7
EC Name:
5-ethylidene-8,9,10-trinorborn-2-ene
Cas Number:
16219-75-3
Molecular formula:
C9H12
IUPAC Name:
5-ethylidenebicyclo[2.2.1]hept-2-ene
Details on test material:
Test substance: 5-Ethylidene-2-norbornene (CAS No. 16219-75-3)
Source: SAN-PETROCHEMICALS Co., LTD., Lot No.7K03
Purity: 99.5%
Stability during uses is confirmed by gas chromatography.

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 9 week old for both sexes
- Weight at study initiation: 320-356 g for males , 203-237 g for females
- Housing: polycarbonate cages spread with Beta chip bedding, individually, in pairs during mating or in litters during lactation period.
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet (e.g. ad libitum): Rat chow pellets available ad libitum.
- Water (e.g. ad libitum): filtered tap water available ad libitum.
- Acclimation period: Animals were quarantined and acclimated for 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22±20°C
- Humidity: at  and 55±15% 
- Air changes: 12 air changes per hour
- Photoperiod: 12 hr light-dark cycle

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- Test material was prepared in corn oil, stored under refrigeration protected from light and used for dosing within 8 days of preparation.  Stability and concentrations of dosing solutions were verified prior to dosing (Analytical method not specified).
- Dosing schedules and pre and post dosing observations periods for P, F1 and F2, if appropriate: Male rats were dosed orally by gavage for 45 days, from 14 days prior to mating, during mating and up to the day before necropsy.  Females were dosed from 14 days prior to mating, through gestation to day 3 of lactation.  
Details on mating procedure:
After 14 days dosing
- M/F ratio per cage: 1:1
- Length of cohabitation: 5 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy

Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Premating exposure period (males): 14 days
Premating exposure period (females): 14 days
Duration of test: maximum 45 consecutive days
Frequency of treatment:
once daily, in the morning
Doses / concentrationsopen allclose all
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:
12
Control animals:
other: yes, corn oil
Details on study design:
dosing volume 5mL/kg

Examinations

Parental animals: Observations and examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: P and F1 Clinical observations performed and frequency: Observations for mortality, external appearance and behavior were made every day before and after dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the day dosing started, on days 3, 7, 14, and weekly thereafter.  Mated females were weighed on days 0, 7, 14, and 20 of gestation, and on days 0 and 4 of lactation. In females, body weight gain during premating, gestation and lactation was calculated from body weight at initiation of dosing, day 0 of gestation, and day 0 of lactation, respectively. 

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption was measured on the day animals were weighed, except during mating.
Oestrous cyclicity (parental animals):
Yes, details not specified.
Sperm parameters (parental animals):
Not performed.
Litter observations:
STANDARDISATION OF LITTERS
- Not applicable.

PARAMETERS EXAMINED
Neonates were examined at parturition to determine number of offspring born (live-births and stillborn), sex, and external anomalies, and observed daily until day 4 of lactation for general condition and mortality. Surviving offspring were weighed individually on days 0 and 4 of lactation, body wt gain was calculated from day 0 body wt.  
Postmortem examinations (parental animals):
SACRIFICE
- Maternal animals: Parameters calculated were gestational period, delivery (#females with live births÷# females which conceived), implantation (#implantation sites÷corpora lutea) and gestation (total # offspring÷#implantation sites) indices.  

GROSS NECROPSY
- Gross necropsy consisted of: Ovaries and uterus of maternal rats were removed at necropsy on day 4 of lactation; and the number of corpora lutea and implantation sites counted.  
Postmortem examinations (offspring):
GROSS NECROPSY
- Offspring were killed on lactation day 4 and necropsied.
- F1 and F2: After the final day of dosing, all surviving rats of both sexes were killed and necropsied.  Livers of males and females, testes and epididymides were weighed and relative organ/body wt at necropsy calculated.  Seminal vesicles and prostate, ovaries, uterus and vagina, and mammary glands from maternal animals in which all neonates died, and sites of macroscopic anomalies were also fixed and retained.  Slides from livers (both sexes), testes and epididymides from control and high dose groups, sites of any anomalies, and ovaries from non-pregnant females, were prepared, stained with hematoxylin-eosin and examined microscopically. Livers from males at the 2 lower dose groups and additional lipid (Oil-Red-O) stained sections from selected high dose males were also examined.
Statistics:
Bartlett's test for homogeneity of variance, ANOVA if variance homogenous or Kruskal Wallis if variance heterogeneous or data non-parametric. Dunnett's or Dunnett's multiple comparison test. Histopathology analyzed by Chi square, then Armitage. Other counted data anlayzed by Fisher's exact probability. maternal animal is specimen unit for neonate data.
Reproductive indices:
Parameters recorded were number of days required for successful mating, number of estrus cycles missed until successful mating, mating index and fertility index. Only 10/12 mated females in the 20 mg/kg group and 11/12 females in the 100 mg/kg were pregnant but fertility indices were not statistically different from controls.
Offspring viability indices:
Live birth index and neonatal viability index on day 4 of lactation were calculated. The live birth index was 100% and lactation behavior was normal in all groups. All pups from one 20mg/kg dam died on day 2 of lactation.  This event did not appear treatment related since no other instances of 100% mortality occurred in other treated groups.  The number of surviving offspring on day 4 of lactation in the high dose group (6.8 pups) was significantly lower than controls (14.2 pups) (p<0.01) but the neonatal viability index on day 4 of lactation (# live pups on day 4 ÷ # live pups on day 0) was not statistically significantly different for any treated group compared to controls.   There were no changes in sex ratio, offspring body wt, appearance of offspring or necropsy findings attributable to test substance.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
not specified
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Two males in the high dose group died 25 days and 36 days after start of dosing. Based on histopathological findings (see below), the deaths may have been as a result of mis-dosing rather than treatment.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body wt gain was suppressed in both sexes in 100 mg/kg group from 7 days after dose initiation to necropsy, but, though a consistent trend, this effect was not always statistically significant. Absolute body wt. in high dose males and females were statistically significantly lower than controls at necropsy (p<0.01).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption decreased in both sexes in 100 mg/kg group from 7 days after dose initiation to necropsy, but, though a consistent trend, this effect was not always statistically significant.
Food efficiency:
not examined
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:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathologic examination of livers from high dose males revealed hypertrophy and vacuolization of hepatocytes; liver sections from males at the 2 lower doses were comparable to controls. Examination of the 2 dead high dose male rats revealed pulmonary and hepatic congestion in both animals and milky appearance and petechiae in the thymus of 1 rat, with histopathologic findings of diffuse thymic hemorrage in that rat, and pulmonary congestion and edema in both rats. (Reviewer's comment - Pulmonary congestion and edema is suggestive of misdosing as a cause of death).

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Number of estrus cycles missed until mating were comparable for treated animals and controls.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
All animals mated within 5 days. Mating index and days required for successful mating were comparable for treated animals and controls.. The gestational period in the 100 mg/kg group (23.1 days) was significantly longer than the control (p<0.01) but was within the normal historical range for this laboratory. Parturition was normal and corpora lutea counts were comparable for all groups. In the 100 mg/kg group, the implantation index (72.8%), total number of offspring born (7.1 pups) and the delivery index (53.1%) were significantly lower than controls (p<0.01), (94.5%, 14.3 pups, and 93.1 %, respectively).

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
histopathology: non-neoplastic

Target system / organ toxicity (P0)

open allclose all
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (nominal)
System:
male reproductive system
Organ:
testes
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Results: F1 generation

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
20 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

NOAEL (NOEL) repeat dose toxicity = 20 mg/kg/day (both sexes)

NOAEL (NOEL) reproduction = 20 mg/kg/day (dam and offspring)

Applicant's summary and conclusion

Conclusions:
5-Ethylidene-2-norbornene administered to rats by oral gavage for 45 days, induced both parental systemic and reproduction/developmental toxic ty at 100 mg/kg/day. Treatment did not appear to affect mating capabilities or fertility index but did affect the embryo and fetus at this high dose t at inhibited body wt gain and food consumption in parental animals. However, the lack of effect on the live birth index, day 4 neonatal viability, exter al appearance and necropsy findings in offspring suggests that the test material does not affect postpartum neonatal growth.
Executive summary:

MHW, Japan (1999) reported results of a guideline (OECD 421) and GLP reproductive/developmental toxicity screening study in Sprague-Dawley rats at doses of 0, 4, 20, and 100 mg/kg/day. ENB was administered for 46 days in males and from 14 days before mating to day 4 of lactation in female rats. Two male rats in the 100 mg/kg group died. Suppressed body weight gain and decreased food consumption was observed in both sexes in this group. The relative liver weights were increased in the 100 mg/kg male group, and histopathological examination revealed hypertrophy and vacuolation of hepatocytes in these animals. Among dams, prolongation of the gestation period, and a trend for a decrease in the number of implantation and delivery indices were observed in the 100 mg/kg group. No other changes attributable to the compound were observed in any parameters including the mating index, the fertility index, the gestation index, number of corpora lutea, parturition state and lactation behavior. Among offspring, total number of births and number of live offspring on day 4 of lactation were decreased in the 100 mg/kg group. No other changes attributable to the compound were observed in parameters including the sex ratio, the live birth index, the viability index on day 4, necropsy findings or external examination. The NOAEL for parental toxicity and for reproductive and developmental toxicity is considered to be 20 mg/kg/day.