N-(2-ethylhexyl)-8,9,10-trinorborn-5-ene-2,3-dicarboximide

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1986-1987

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1987

Report date:

1987

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EPA OPP 83-3 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N-(2-ethylhexyl)-8,9,10-trinorborn-5-ene-2,3-dicarboximide

EC Number:

204-029-1

EC Name:

N-(2-ethylhexyl)-8,9,10-trinorborn-5-ene-2,3-dicarboximide

Cas Number:

113-48-4

Molecular formula:

C17H25NO2

IUPAC Name:

N-(2-ethylhexyl)-8,9,10-trinorborn-5-ene-2,3-dicarboximide

impurity 1

Reference substance name:

(E)-1,4-Bis(2-ethylhexylamino)-2-butene-1,4-dione

Molecular formula:

C20H38N2O2

IUPAC Name:

(E)-1,4-Bis(2-ethylhexylamino)-2-butene-1,4-dione

impurity 2

Reference substance name:

1-(2-ethylhexyl)-1H-pyrrole-2,5-dione

Molecular formula:

C12H19NO2

IUPAC Name:

1-(2-ethylhexyl)-1H-pyrrole-2,5-dione

Test material form:

liquid

Details on test material:

Purity and characterisation analysis conducted on the following sample; Supplier: McLaughlin Gormley King ; Batch Number: AB9500

Specific details on test material used for the study:

MGK 264
LOT 3843
Active Ingredients: N - Octybicycloheptene dicarboximide 98.00%

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Eighty-five untreated, sexually mature 4 month old, virgin female New Zealand White SPF rabbits were received from Hazleton Research Animals, Denver, Pennsylvania on August 13, 1986. During the 142-day acclimation period, the animals were carefully observed for
changes in appearance and behavior. each animal was provided with basal Rabbit Chow® #5322 and tap water ad libitum.

The temp/humidity recording was monitored and maintenance was notified if protocol-designated ranges (temperature 15 to 19°C, humidity 30 to 70%) were exceeded. Fluorescent lighting provided illumination 12 hours per day. The females were housed individually in suspended stainless steel cages from receipt until sacrifice. Nesting material was not provided since the females were sacrificed prior to delivery. Each female was identified by cage,"group and individually by a Monel® metal ear tag bearing its animal number. The individual animal number plus the IRDC study number comprised a unique identification number for each animal. The females were approximately 8-1/2 months old at the time of insemination and weighed between 3672 and 5452 g on gestation day 0.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5% methylcellulose

Details on exposure:

The test article was prepared at concentrations to permit the administration of dose levels of 10, 30 and 100 mg/kg/day at a dosage volume of 3 ml/kg. The test article was administered as a single daily dose on days 7 through 19 of gestation. Test article administration was accomplished by intragastric intubation using 20 cc disposable plastic syringes and 12 gauge, 15 cm long curved stainless steel dosing needles. Dosing began approximately 6 to 10 1/4 hours after initiation of the illuminated phase of the photoperiod.
The control group received the vehicle only on a comparable regimen at a volume of 3 ml/kg. Individual dosages were determined from the most recently recorded individual body weights.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

During the study, the 24-hour stability of test suspensions was assessed. In addition, the first daily preparation of each dosing week was analyzed for concentration.
Gas Chromatograph with FID detector used:
Gas Chromatograph Parameters (may be changed as necessary to optimize analyte chromatography) .
1. Instrument; Tracor 560 fitted with flame ionization detector (FID)
2. Column; 6' x 4 mm glass column packed with 5% OV-101 on chromosorb W/HP
3. Temperatures; Oven 157°C, Detector 283°C Injector 242°C
4. Flow rate; Carrier; (Nz) 27 ml/min, Air; 1. 45 SCFH, Hz; 30 mg/min
5. Injection volume; about 6 mcl.
Inject equal volumes of standard and sample in the order; standard, sample, sample, standard, sample, etc ...
Measure peak heights of the internal standard and the first component of the 2 component test article.

Details on mating procedure:

Eight proven male rabbits of the same breed and source were selected to serve as donors. Semen was collected using an artificial vagina and the gelatinous plug was removed from the ejaculate. The semen was immediately evaluated for motility and was used for insemination only if the motility was 60% or greater. The ejaculate was diluted with 3.0 ml of 0.9% Sodium Chloride for Injection U.S.P. at 33-37°C and 0.3 ml of this dilute semen was introduced into the anterior vagina of the female using an insemination pipette. A minimum of 0.5 million sperm/doe was provided at insemination. Immediately after insemination, ovulation was induced by an injection of 100 U.S.P. units of human chorionic gonadotropin into a marginal ear vein. Semen from one male was used to inseminate an equal number of females in each group. Insemination procedures were performed on three consecutive days and an equal number of females were inseminated in each group per day. The day of insemination was designated as day 0 of gestation.

Frequency of treatment:

single daily dose on days 7 through 19 of gestation.

Duration of test:

29 days of gestation

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

10 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

30 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle control group

No. of animals per sex per dose:

16 female rabbits per dose group

Control animals:

yes, concurrent vehicle

Examinations

Maternal examinations:

Survival, Appearance and Behavior
Throughout the study the females were observed twice daily for mortality and overt changes in appearance and behavior. The presence and duration of clinical signs of toxicity were recorded once daily on days 0 through 29 of gestation. Females not surviving to the scheduled sacrifice were necropsied in an attempt to determine the cause of death. Any female showing signs of abortion or premature delivery was sacrificed and necropsied on the day such evidence was observed. Fetuses from does that died, aborted, delivered or were sacrificed within a day of scheduled Cesarean section were processed and evaluated as for the scheduled termination animals. Fetuses from does dying or aborting prior to this time were preserved in neutral buffered 10% formalin for possible future ewaluations. Maternal tissues were preserved in neutral buffered 10% formalin for microscopic examination as deemed necessary by the gross findings.

Ovaries and uterine content:

Immediately following sacrifice, the uterus and ovaries were exposed by an abdominal incision. The number and location of viable and nonviable fetuses, early and late resorptions and the number of total implantations and corpora lutea were recorded. The uterus was then excised and the fetuses removed. The abdominal and thoracic cavities and organs of the does were examined for grossly evident morphological changes and the carcasses discarded. Maternal tissues were preserved in neutral buffered 10% formalin for histopathological examination as deemed necessary by the gross findings. Uteri from females that appeared nongravid were opened and placed in 10% ammonium sulfide solution for detection of implantations.

Fetal examinations:

All fetuses were individually weighed, tagged and examined for external malformations and variations. Each fetus was dissected, internally sexed and examined for visceral malformations and variations, including the brain by a mid-coronal slice. The heart was dissected by a modification of the method described by Staples. The eviscerated skinned fetuses were fixed in alcohol, macerated in potassium hydroxide, stained with Alizarin Red S and cleared with glycerin by a method similar to that described by Dawson for subsequent skeletal examination.

Statistics:

A.ll statistical analyses compared the treatment groups with the control group, with the levels of significance ac p<0.05 and p<0.01. All
means were accompanied by standard deviations. Male to female fecal sex ratios were compared using the Chi-square test criterion with Yates' correction for 2 x 2 contingency tables and the proportions of litters with malformations were compared using Fisher's exact probability test as described by Siegel to determine the significance of differences.
The proportions of resorbed and dead fetuses and post-implantation losses were compared by the Mann-Whitney U-test as described by Siegel and Weil to determine the significance of differences.
Numbers of corpora lutea, total implantations and live fetuses, mean maternal and fecal body weights and maternal body weight changes were compared by analysis of variance (one-way classification), Bartlett's test for homogeneity of variance and the appropriate t-test (for equal or unequal variance) as described by Steel and Torrie using Dunnett's multiple comparison tables to determine the significance of differences.

Historical control data:

The rabbit is an acceptable model for teratology studies. This laboratory has historical control data on the incidence of fetal malformations and variations in this strain from this source. This strain is susceptible to known teratogenic agents.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There was an increased incidence of post-dose excessive salivation at the high-dose level compared to the control group. This finding was considered treatment related. This was also seen in one mid-dose animal.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Control - 1 aborted
10mg/kg - 1 died and 1 aborted
30mg/kg - 3 died and 1 aborted
100mg/kg - 1 aborted
Given the lack of a dose-relationship, the deaths and abortions seen in this study were not considered treatment related

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no biologically relevant differences between the mean maternal body weight changes of the treated groups and those of the
control group during the treatment (gestation days 7 through 19), overall gestation (gestation days 0 to 29) or adjusted gestation (gestation days 0 to 29 less the weight of uterus and contents) intervals.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Maternal developmental toxicity

Number of abortions:

effects observed, non-treatment-related

Description (incidence and severity):

Control - 1 aborted
10mg/kg - 1 died and 1 aborted
30mg/kg - 3 died and 1 aborted
100mg/kg - 1 aborted
Given the lack of a dose-relationship, the deaths and abortions seen in this study were not considered treatment related

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

effects observed, non-treatment-related

Description (incidence and severity):

Only 1 loss seen in study at 100mg/kg therefore considered not treatment related.

Early or late resorptions:

not specified

Dead fetuses:

not specified

Changes in pregnancy duration:

not specified

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified

Changes in number of pregnant:

no effects observed

Other effects:

not examined

Details on maternal toxic effects:

MGK 264 was considered to be responsible for an increase in the incidence of excessive salivation in the high dose females (100 mg/kg bw/day). The high dose in this study was selected based on the findings of a dose range finding study in which there was excessive toxicity at 300 mg/kg bw/day (lowest dose tested).

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

clinical signs

Results (fetuses)

Fetal body weight changes:

not specified

Reduction in number of live offspring:

not specified

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

All malformations of fetuses observed were noted exclusively among the treated groups. However, no relationship to treatment was assumed as the anomalies seen in each group occurred in single incidence. See attached table Fetal Malfunctions (background information)

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

All malformations of fetuses observed were noted exclusively among the treated groups. However, no relationship to treatment was assumed as the
anomalies seen in each group occurred in single incidence. See attached table Fetal Malfunctions (background information)

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

All malformations of fetuses observed were noted exclusively among the treated groups. However, no relationship to treatment was assumed as the anomalies seen in each group occurred in single incidence. See attached table Fetal Malfunctions (background information)

Other effects:

not examined

Effect levels (fetuses)

Key result

Dose descriptor:

NOEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no significant changes observed were caused by test material at 100 mg/kg/day (highest dose level tested)

Overall developmental toxicity

Key result

Developmental effects observed:

no

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

Applicant's summary and conclusion

Conclusions:

Treatment of pregnant New Zealand White SPF rabbits with MGK 264 did not elicit fetotoxic effects at any dosage level tested. The test article was considered responsible for an increase in the incidence of excessive salivation in the maternal high- dose group animals. In conclusion, the no observable effect level of MGK 264 with respect to fetotoxicity was 100 mg/kg/day (high-dose) when administered orally to pregnant New Zealand White SPF rabbits.

Executive summary:

Mated female New Zealand White Rabbits, randomly assigned to one control and three treatment groups of 16 animals each, were used to determine the developmental toxicity potential of MGK® 264. Dosage levels of 10, 30 and 100 mg/kg/day were administered orally by gavage as a single daily dose on days 7 through 19 of gestation at a volume of 3 ml/kg. The control group received the vehicle only, 0.5% methylcellulose, on a comparable regimen. Cesarean sections were performed on all surviving females on gestation day 29 and the fetuses were removed for teratologic evaluation.

 

Treatment of New Zealand White rabbits with MGK® 264 elicited maternally toxic effects (increased incidence of excessive salivation) in the high-dose group. The test article did not induce developmentally toxic effects at any dosage level.

 

In summation, the no observable effect level of MGK® 264 with respect to developmental toxicity was 100 mg/kg/day when administered orally by gavage to New Zealand White Rabbits.