Ethylene dinitrate

Repeated dose toxicity: oral

Administrative data

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

publication

Title:

Subacute and chronic toxicity studies of trinitroglycerin in dogs, rats, and mice

Author:

Ellis III, H.V., Hong, C.B., Lee, C.C., Dacre, J.C. and Glennon, J.P.

Year:

1984

Bibliographic source:

Fundamental and Applied Toxicology, 4(2): 248-260

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 452 (Chronic Toxicity Studies)

Principles of method if other than guideline:

Solubility and hydrolysis characteristics, f.pt., and m.pt. of glycerol trinitrate were not determined for this study. They are published. Food intake was measured weekly for the first four weeks and for one week / month, thereafter. Body weights were measured weekly until the body weight leveled off, and biweekly thereafter. Blood was collected for hematology analyses from tail tips of (where possible) the same four males and four females before the test started, and at the end of 3, 6, 9, 12, 18, and 24 months. Interim blood chemistry analyses were performed at twelve months. There were no urinalyses. Rectum, femur and aorta were not routinely examined microscopically. At the end of 12 months and 24 months, four males and four females from each group were taken off the test diet, put on "control" diets, and maintained as recovery groups under conditions otherwise identical to those of the test and control groups. These two sets of "recovery" rats were sacrificed at the end of months 13 and 25, respectively, and subjected to the same clinical, hematological, macroscopic, and microscopic examinations as the animals sacrificed at 12 and 24 months, those sacrificed in extremis, and the unscheduled deaths. Also, after 12 months on test, abdominal aortal blood was taken for hematology and clinical chemistry from four males and four females from each group. These animals were then sacrificed, necropsied, and their organs examined for histopathologic effects. In addition, four males and four females from each group were scheduled for sacrifice at each of 3, 6, 9, and 18 months for the same 1) blood hematologic and chemical, 2)gross whole-body and organ, and 3) histopathologic examinations. Any remaining survivors at 24 months were scheduled for the same examinations.

GLP compliance:

no

Remarks:

study pre-dated USFDA GLPs

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Glycerol trinitrate

EC Number:

200-240-8

EC Name:

Glycerol trinitrate

Cas Number:

55-63-0

Molecular formula:

C3H5N3O9

IUPAC Name:

propane-1,2,3-triyl trinitrate

Test animals

Species:

rat

Strain:

other: Charles River CD

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratory, Wilmington, Massachusetts, USA
- Age at study initiation: All animals were maturing
- Fasting period before study: no data
- Housing: plastic cages with metal lids, filter tops, 4 male or 5 female in each cage, some groups were subdivided to prevent fighting.
- Identification of animals: ear-punches
- Bedding: with hardwood chip
- Diet: ad libitum, Diets were prepared weekly
- Water: ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 75+/-5
- Humidity (%): 50+/-10%
- Air changes (per hr): 10 air changes per hour
- Photoperiod (hrs dark / hrs light):12 hour light/12 hour dark

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION:
- Rate of preparation of diet (frequency): Diets were prepared weekly.
- Mixing appropriate amounts with (Type of food): 10% concentrate was mixed with feed in a rotating box on a modified mixer to provide the diet mixture by successive dilutions. The control rodents received a mixture containing 10% dried feed in ordinary feed. With the rodents, dosage levels of 0,01% (100 ppm), 0,1% (1000 ppm) and 1% (10 000 ppm) in the diet were used.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

24 hours/day (continuous) for two years

Frequency of treatment:

daily in the diet

Doses / concentrations

Remarks:

Doses / Concentrations: 0.0, 0.01, 0.1 1.0% (w/w) of diet. Corresponds to male and female daily intakes of 0.0, 3.04 ± 0.16, 31.5 ± 1.6, 363 ± 10 and 3.99 ±0.18, 38.1 ±1.6, and 434 ± 11 mg NG / kg day, respectively (means ± standard errors of 24 monthly measurements). Basis:actual ingested

No. of animals per sex per dose:

The begining number of rats was 38 of each sex per group.

Control animals:

yes

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: YES
- Time schedule: daily
- Cage side observations were included: behavioral changes

DETAILED CLINICAL OBSERVATIONS: YES
- Time schedule: daily
- All animals were observed for toxic signs.

BODY WEIGHT: YES
- Time schedule for examinations: weekly for the first 6 months, after weight gain leveled off, they were weighed biweekly.

FOOD CONSUMPTION AND COMPOUND INTAKE: YES
- Food consumption was measured during the first 4 weeks and then during the last week of each month.

HAEMATOLOGY: YES
- Time schedule for collection of blood: After 12 months dosing
- Number of animals: 4 males/4 females from each dosage group were bled from their aortas.
- Parameters examined: erythrocyte, reticulocyte, leucocyte, and platelet counts; hematocrit, hemoglobin, erythrocyte indices, methemoglobin, Heinz bodies, and c lotting time.

CLINICAL CHEMISTRY: YES
- Time schedule for collection of blood: After 12 months dosing
- Number of animals: 4 males/4 females from each dosage group
- Parameters examined: fasting blood glucose, serum SGOT, serum SGPT, APase, and BUN. Special tests, e.g., serum electrolytes were to be performed whenever indicated

Sacrifice and pathology:

GROSS PATHOLOGY: YES

ORGAN AND OTHER WEIGHTS: Brain, heart, liver, kidneys, spleen and gonads.

HISTOPATHOLOGY: YES

Statistics:

In general, standard methods (Steel & Torrie, 1960), with p<0.05 considered significant. Continuous variables were analysed by Dunnett'smultiple comparison procedure after an analysis of variance or Student's t test. Enumeration data, such as tumor incidence, were analysed by Fisher's exact probability test. In some of the histopathologic incidence analyses the CHI square test or exact probabilities on contingency tables with p<0.05 were considered significant.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Haematological findings:

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Details on results:

CLINICAL SIGNS AND MORTALITY: Rough fur, matted appearance

BEHAVIOUR: A lack of grooming

BODY WEIGHT AND WEIGHT GAIN: High dose: depressed weight gain

FOOD CONSUMPTION: High dose: decrease feed consuption

HAEMATOLOGY: High dose: methemoglobinemia, elevated erythrocyte, hematocrit and hemoglobinSmall amounts of methemoglobin were occasionally seen in other dose groups.

CLINICAL CHEMISTRY: Abnormal values for glucose, alkaline phosphate, SGOT, SGPT

ORGAN WEIGHTS: Remarkably large livers
High dose: high kidney weights were normal variation, increase in spleen weights were normal variation

GROSS PATHOLOGY AND HISTOPATHOLOGY:
High dose:
- Liver: 1) cholangiofibrosis, proliferace of the bile ducts, fibrous tissue, white patches of various sites, cystic, adenomatoid, 2) development of hepatocellular carcinoma, occasional metatatstic nodules in the lungs
- Testis: Cell tumors in the testis (pressure on the tubules, aspermatogeneisis)
- Spleen and renal epithelium: Pigment deposit (hemosiderin)
The most common tumors in the high dose rats: Pituitary adenoma (males/females), Mammary (females), Primarily fibroadenomas (females).
These tumors were the most common causes of death.

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

3.04 mg/kg bw/day (actual dose received)

Sex:

male

Basis for effect level:

other: The low dose ( 24 month ave. in males = 3.04 ± 0.16 mg glycerol trinitrate / kg /day; in was a "no-effect" dose in males by any of the criteria used.

2

Dose descriptor:

NOAEL

Effect level:

3.99 mg/kg bw/day (actual dose received)

Sex:

female

Basis for effect level:

other: The low dose ( 24 month ave. in females = 3.99 ± 0.18 mg glycerol trinitrate / kg / day) was a "no-effect" dose in femalesby any of the criteria used.

3

Dose descriptor:

LOAEL

Effect level:

31.5 mg/kg bw/day (actual dose received)

Sex:

male

Basis for effect level:

other: The middle dose (24 month ave. in males = 31.5 ± 1.6 mg glycerol trinitrate / kg / day was an effect dose in males.

4

Dose descriptor:

LOAEL

Effect level:

38.1 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: The middle dose (24 month ave. in females = 38.1 ± 1.6 mg glycerol trinitrate / kg / day) was an effect dose in females.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The high dose with glycerol trinitrate intake of 363 mg/kg/day in males and 434 mg/kg/day in females was toxic, decreased feed consumption and depressed weight gain. The target organs included the blood (methemoglobin), liver (cholangiofibrosis and hepatocellular carcinoma) and testis (intersticial cell tumor). There was a decrease in the naturally occurring tumors of the pituitary and mammary gland, which decreased the death rate in females.