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

Repeated dose toxicity: oral

Administrative data

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 1989 to December 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EPA OPP 83-1 (Chronic Toxicity)

GLP compliance:

yes

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 7437, pale yellow viscous liquid was received at the experimental laboratory on 10/04/1988 in 2 drums. The test material was provded by the Sponsor. The test material was stored at rom temperature under environmentally controlled conditions. Samples of the test material were collected at 6 month intervals and sent back to the sponsor for analysis.

Test animals

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

Twenty (20) male and 20 female purebred beagle dogs (approximately
3 1/2 to 4 1/2 months of age) were obtained from Ridglan
Farms, Inc., Mount Horeb, Wisconsin on September 13, 1989. A few of
the animals were slightly younger than the 4 months of age required
by the protocol at receipt. In the opinion of the Study Director
this protocol deviation did not affect the quality or the intgrity
of the study. The dogs were immunized against distemper, parvovirus,
parainfluenza, adenovirus type 2, Bordetella, leptospirosis
and rabies by the breeder prior to receipt at IRDC. Flotation tests
were conducted on stool samples obtained from each dog; the results
of the tests were negative.

At various intervals during the study, otitis was
noted for a few dogs in the control and treated groups; the appropriate
treatments were performed. Clinical pathology and ophthalmological
examinations were conducted on each dog prior to study
initiation. From these animals, sixteen males (weighing from 8.9 to
14.8 kg) and sixteen females (weighing from 9.1 to 11.8 kg) were selected
on the basis of best physical condition. The 16 dogs of each
sex were assigned at random to control or treated groups.
The dogs were individually housed in stainless steel cages
equipped with automatic watering valves and maintained in an environmentally
controlled room with a 12 hour light/12 hour dark cycle.
The temperature and humidity were continuously recorded and the recordings
were monitored for conformance with protocol specifications.
Appropriate corrective action was taken whenever readings
were outside acceptable limits. On some occasions the results of
the environmental monitoring and/or documentation of corrective
action(s) were inadvertently not recorded. In the opinion of the
Study Director, these protocol deviations did not affect the quality
or the integrity of the study. The graphs of temperature and humidity
are maintained in the study records.

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

A potential route of administration to humans is oral.
Therefore, this study employed the oral route.

Vehicle:

other: Premixed with Certified Canine Diet #5007, Purina Mills, Inc., St. Louis, Missouri

Details on oral exposure:

Route: via diet
Frequency of Administration: continuously in the diet

Preparation of Diet Mixtures: at least once weekly
Storage of Diet Mixtures: room temperature
Diet Mixtures Stable for a Period of: 21 days

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For Determination of Test Article Concentration throughout the study, 100 gram samples were taken from
the top, middle and bottom of each diet at the time test article:diet mixtures were prepared. The three samples (top, middle and bottom) were mixed to form a composite sample, which was then subdivided into three 100 gram samples. For study weeks 1-4 and every
four weeks thereafter, one set of these triplicate samples was analyzed in duplicate for determination of test article concentration
Duplicate 10g subsamples were extracted by petroleum ether using Soxhlet equipment and dose concentrations quantified by GC. The other two sets were stored frozen. At all other study weeks all three sets of samples were stored frozen.

Duration of treatment / exposure:

MGK® 264 was offered in the diet to beagle dogs at constant concentrations of 65, 250 and 1,000 ppm for
one year; a control group received ground diet on the same regimen.

Frequency of treatment:

food available ad libitum, except prior to clinical pathology testing and necropsy.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

65 ppm

Doses / concentrations 2

Dose / conc.:

250 ppm

Doses / concentrations 3

Dose / conc.:

1 000 ppm

No. of animals per sex per dose:

4 female/ 4 male

Control animals:

yes, plain diet

Details on study design:

MGK® 264 was offered in the diet to beagle dogs at constant concentrations of 65, 250 and 1,000 ppm for
one year; a control group received ground diet on the same regimen. Four male and four female dogs were initiated on study for each of the four groups.

Examinations

Observations and examinations performed and frequency:

Mortality, moribundity and overt toxicity at least twice daily; detailed observations at least once each.
Bodyweights determined pre-test and weekly thereafter.
Food consumption determined weekly.
Ophthalmoscopic examinations carried out pretest, 12, 24 and 51 weeks of study.

On all dogs pretest and at 6, 12, 25 and 50 weeks of study:
Hematocrit; hemoglobin; erythrocyte count; mean corpuscular hemoglobin (MCH), volume (MCV), and hemoglobin concentration (MCHC); total and differential leukocyte counts; platelets; reticulocytes.
Aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, lactate dehydrogenase, leucine aminopeptidase, glucose, urea nitrogen, total bilirubin, cholesterol, albumin, globulin (calculated), albumin/globulin ratio (calculated), total prote1n,
creatinine, inorganic phosphate, creatine phosphokinase, sodium, potassium, chloride, calcium. Volume, color and appearance, pH, specific gravity, protein, glucose, ketones, urobilinogen, nitrite, bilirubin, leucocytes, occult blood, microscopic element.

Sacrifice and pathology:

Anatomic pathology on all surviving animals at study week 52. Tissues fixed in formalin (except eye, in a glutaraldehyde fixative, and testis and epididymis in Bouin's_ fixative). Bone marrow smears collected at.terminal sacrifice. Organ weights taken for all dogs sacrificed at study termination. Absolute and relative (to body and brain weights) weights of liver, kidney (2), heart, spleen, testis (2), brain, ovary (2), pituitary, thyroid/parathyroid (2), adrenal (2). Tissues examined: Adrenal (2), aorta, bone (rib), bone marrow (rib), brain (fore, mid and hind), eye including optic nerve (2), gallbladder, esophagus, stomach (glandular and nonglandular), duodenum,
jejunum, ileum, cecum, colon, rectum, ovary (2), testis with epididymis (2), heart, kidney (2), liver (3 sections), lung with mainstem bronchi (2), lymph nodes (tracheobronchial, mesenteric), mammary gland (female only), pancreas, pituitary, prostate, salivary gland
(mandibular with mandibular lymph node), sciatic nerve, skeletal muscle (thigh), skin, spinal cord (cervical, (mid)thoracic and lumbar), spleen, thymus, thyroid/parathyroid (2), trachea, urinary bladder, uterus, vagina, gross lesions, animal identification site.
Microscopic pathology examined on all dogs. Tissues sectioned at 5 microns and stained with hematoxylin and eosin.

Statistics:

Body weights, food consumption values, clinical pathology parameters and organ weights analyzed using analysis of variance and Bartlett's test. Treatment groups compared to the control group, by sex, using the
appropriate t-statistic (equal or unequal variance). Nonparametric analysis, when appropriate, by rank transformation.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No adverse effects were found during the study. In general animals receiving MGK 264 gained more weight than control animals especially in male animals.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

In general, the g/animal/day food consumption of the dogs in the treated groups was increased compared to that of the control
dogs, with the difference statistically significant in isolated instances.
However, these increases did not occur in a dose-related pattern. Similarly, the g/kg/day food consumption of the treated females was increased, although not in a dose-related fashion, during the first 6 months of the study. During the latter half of the study, the values for this parameter were comparable for the control and treated females. The q/kg/day food consumption of the treated males fluctuated in relation to the control values; no dose-related or statistically significant differences were observed.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance related organ weight variations were discerned in any of the experimental groups. A number of statistically significant variations occurred but these were considered to reflect normal biological variation because there was no dose response or correlation with any morphologic changes.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test substance related microscopic findings were limited to the liver of male and female dogs from the 1,000 ppm group. Brown pigment considered to represent biliary stasis, because of its apparent intracanalicular location and its deep, reddish brown color (which was characteristic of bile) was present for the majority of the dogs in this group. Two male dogs from the 1,000 ppm group also had
trace hepatocellular hypertrophy which was also considered test substance related.Note that these findings in the liver were not associated with degenerative change.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 ppm

Based on:

other: MGK 264 in the diet equivalent to 34.9/32.5 mg/kg bw/day in males/females

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Key result

Critical effects observed:

not specified

Lowest effective dose / conc.:

1 000 ppm

System:

hepatobiliary

Organ:

liver

Applicant's summary and conclusion

Conclusions:

In summation, MGK® 264 was offered in the diet to beagle dogs at constant concentrations of 65, 250 and 1,000 ppm for one year; a control group received ground diet on the same regimen.
Four male and four female dogs were initiated on study for each of the four groups.
MGK® 264 induced microscopic changes of brown pigment in the liver and trace hepatocellular hypertrophy when offered in the diet at a concentration of 1,000 ppm to beagle dogs for one year. No other changes were observed that could be related to treatment with the test substance. The no-observed-effect-level (NOEL) dose concentration was considered to be 250 ppm in the diet which was equivalent to 7.5 mg/kg bw/day in males and 7.4 mg/kg bw/day in females based on average compound consumption.

However, these changes suggested that the liver was involved in the metabolism and excretion of the test substance. Trace hepatocellular hypertrophy and cytoplasmic pigment in some animals reflected the additional work load as a consequence of ingestion of large quantities of test substance. Degenerative changes were not identified. Therefore, in the absence of degenerative changes occurring in the liver the systemic no-observed-adverse-effect-level (NOAEL) is 1000 ppm equivalent to 34.9/32.5 mg/kg bw/day in males/females.

Executive summary:

A one year dietary administration study of MGK 264 was performed in dogs according to EPA OPP 83 -1 (chronic toxicity).

MGK® 264 was offered in the diet to beagle dogs at constant concentrations of 65, 250, and l,000 ppm for one year. A control group received ground diet on the same regimen. During the study twice daily observations of mortality/moribundity and overt toxicity were performed. The dogs were weighed weekly and the food consumed was measured weekly. Ophthalmoscopic examinations of the eyes were performed pre-trial and again at weeks 12, 24 and 51. Clinical pathology examinations were performed pre-trial and at weeks , 12, 25 and 50. At the end of the treatment period the animals were sacrificed and necropsy examinations performed. Selected tissues were weighed and a full list of tissues processed for microscopic examination.

All dogs survived the treatment period. No adverse effects were recorded for clinical signs, body weight, food intake, clinical pathology, macroscopic findings or organ weight. Microscopic findings of cytoplasmic pigment possibly indicating biliary stasis, was noted in the livers of the 1000 ppm males and females. In addition there was trace hepatocellular hypertrophy in 2 of the 4 males from this group. None of these changes were associated with degenerative conditions.

On the basis of the findings in the liver, the NOEL was considered to be 250 ppm which is equivalent to 7.5 mg/kg bw/day in males and 7.4 mg/kg bw/day in females based on average compound consumption. However, these changes suggested that the liver was involved in the metabolism and excretion of the test substance.Trace hepatocellular hypertrophy and cytoplasmic pigment in some animals reflected the additional work load as a consequence of ingestion of large quantities of test substance.Degenerative changes were not identified.Therefore, in the absence of degenerative changes occurring in the liver the systemic no-observed-adverse-effect-level (NOAEL) is 1000 ppm equivalent to 34.9/32.5 mg/kg bw/day in males/females.