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Diss Factsheets

Toxicological information

Repeated dose toxicity: oral

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Administrative data

chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to Guideline study with acceptable restrictions.

Data source

Referenceopen allclose all

Reference Type:
study report
Report date:
Principles and Procedures of Statistics.
Steel, R.C.D. and J.H. Torrie
Bibliographic source:
McGraw-Hill Book Co., New York.

Materials and methods

Test guideline
equivalent or similar to guideline
OECD Guideline 452 (Chronic Toxicity Studies)
Principles of method if other than guideline:
Solubility and hydrolysis character-istics,, and of NG were not determined for this study. Blood (jugular) clinical chemistry and hematology analyses were performed at zero, three, six, nine, and twelve months. Three males and three females were sacrificed from each group after twelve months. There was a one month recovery period for the remaining three males and free females in each group. Total protein and albumin were not determined in the blood samples. There were no urinalyses. Rectum, femur, and aorta were not routinely examined microscopically. In addition to the organs called for in OECD Protocol #452, diaphragm, tongue, tonsils, trachea, and ureter routinely were removed, fixed, and examined microscopically. At the end of the recovery period, blood samples were taken from the recovery dogs for selected hematology measurements. The recovery dogs were then sacrificed and organs were removed, processed, sectioned, and examined microscopically as described above.
GLP compliance:
study pre-dated even USFDA GLPs.
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
Glycerol trinitrate
EC Number:
EC Name:
Glycerol trinitrate
Cas Number:
Molecular formula:
C3H5N3O9 C3H5(NO3)3
propane-1,2,3-triyl trinitrate
Details on test material:
- Name of test material (as cited in study report): Nitroglycerin, TNG
- Substance type: technical product
- Physical state: a 10 % mixture on lactose
- Analytical purity: 9.72% +/- 0.09%
- Lot/batch No.: D17-H3
- Source: Atlas Chemical Division, ICI America Inc., Wilmington, DE

Test animals

Details on test animals or test system and environmental conditions:
TEST ANIMALS- Source: Hazleton Research Animals (Cumberland, Virginia)- Housing: dog pens with outside runs. Up to 12 dogs shared 60 sq ft heated inside space and 120 sq ft of outside space.- Diet (e.g. ad libitum): ad libitum- Water (e.g. ad libitum): continuously-ad libitum- Acclimation period: at least 2 weeks

Administration / exposure

Route of administration:
oral: capsule
other: lactose
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Amount of test material required for the doses were adjusted weekly, based on weekly weight of each dog. The dogs receiving the 5 mg / kg dose received one capsule daily, containing the appropriate amount of the 9.72 % concentration of TNG on lactose. Dogs receiving the 25 mg / kg dose received half the appropriate amount of the same formulation in each of two sequentially administered capsules. For the dogs receiving the 1 mg / kg / dose, the TNG / lactose mixture was further diluted with lactose to 2% TNG and they were given their daily dose in one capsule containing this diluted TNG / lactose.
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
The supplied sample was studied by gas chromatography (content of TNG in capsule).
Duration of treatment / exposure:
12 months
Frequency of treatment:
Doses / concentrations
Doses / Concentrations:1, 5, 25 mg TNG/kg/dayBasis:actual ingested
No. of animals per sex per dose:
6 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for animal assignment: Each animal is assigned a four-digit number. The first two digits indicate the dosage groups for TNG, i.e., 80, 81, 82 and 83 for the control, low, middle or high dose group, respectively. The last one or two digits are the animal numbers within each species.- Schedule: After 12 months dosing, three male and three female dogs in each group were killed for necropsy. The treatment of the remaining dogs in each group was discontinued for 4 weeks. These dogs were used on a recovery study and killed for necropsy at the end of 13 months.


Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes - Time schedule: dailyDETAILED CLINICAL OBSERVATIONS: Yes - Time schedule: dailyBODY WEIGHT: Yes - Time schedule for examinations: weeklyFOOD CONSUMPTION: - Time schedule: 1 week each month- The feed consumption was measured by placing dogs in a metabolism cage, giving them a measured amount of feed, waiting 0,5 hr, then returning them to their pen and estimating the remaining amount of feed by volume. This value was converted to weight by a factor determined by averaging the weight of 20 replicates of volume measurements.HAEMATOLOGY: Yes - Time schedule for collection of blood: before dosing and at the end of 3, 6, 9 and 12 months during dosing- Anaesthetic used for blood collection: No data- Animals fasted: No data- Parameters examined: erythrocyte, reticulocyte, leucocyte and platelet counts, hemacrit, hemoglobin, erythrocyte indices, methemoglobin, Heinz bodies and clotting time.CLINICAL CHEMISTRY: Yes - Time schedule for collection of blood: before dosing and at the end of 3, 6, 9 and 12 months during dosing- Animals fasted: No data- Parameters examined: fasting blood glucose, serum glutamic-oxaloacetic transaminase, serum glutamic-pyruvic transaminase, alkaline phosphatase and blood urea nitrogen.URINALYSIS: Yes- Time schedule for collection of urine: before dosing and at the end of 3, 6, 9 and 12 months during dosing- Metabolism cages used for collection of urine: Yes - Animals fasted: No- Parameters examined: protein, sugar, microscopic examinationOTHER:- Immunoglobin E - Occult Blood in Feces
Sacrifice and pathology:
In general, standard methods (Steel & Torrie, 1960) with p<0.05 considered significant. Continuous variables were analyzed by Dunnett's multiple comparison procedure after an analysis of variance, or Student's t test. Enumeration data, such as tumor incidence, were analyzed by Fisher's exact probability test. In some of the histopathological incidence analyses the CHI square test or exact probabilities on contingency tables were used with p<0.05 considered significant.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Details on results:
HAEMATOLOGYToxicologically important finding was small amounts of methemoglobin in some dogs treated for 6 months or longer. In almost all cases, the amount found was very small (less than 3%). Because the assay method involves a differance in absorption values (before and after conversion of methemoglobin to cyanmethemoglobin), a small apparent value could be an artifact. After 6 months, there was a scattered incidence in all groups, this is not significant. But after 9 months, there was methemoglobin in half or more of the low-dose males (given 1 mg/kg/day) and middle-dose (5 mg/kg/day) males and females, and all but one of the high-dose (25 mg/kg/day) dogs. The trend is significant in both sexes, although only the data in males are statistically significant. After 12 months, the dose response in incidence was not apparent. However, the high-dose male (5.3%) and one of the high-dose females (3.7%) had the highest values of methemoglobin seen in the entire studyORGAN WEIGHTSStatistically significant , but toxicologically not important, differences were seen in the pituitary weights of middle-dose males and the heart weights (relative to brain) of high-dose males.GROSS PATHOLOGYGross pathology, such as lung nodules, correlated well with the histopathologic lesions. None of the lesions, whether common (mild hepatocytic vesiculation) or rare (severe lymphocytic thyroiditis in No. 83-37 or the mild endometritis in No. 82-26) correlated with TNG treatment.

Effect levels

open allclose all
Dose descriptor:
Basis for effect level:
other: Under conditions of this study, there was no NOAEL, in either sex.
Remarks on result:
not measured/tested
Effect level not specified (migrated information)
Dose descriptor:
Effect level:
1 mg/kg bw/day (actual dose received)
Basis for effect level:

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

The only effect that could be attributed to TNG in this study was a mild methemoglobinemia that was not accompanied by any of the sequelae of a severe methemoglobinemia such as Heinz bodies, elevated reticulocytes, and anemia. Except for the dogs receiving 25 mg TNG / kg / day, methemoglobin levels returned to zero after a thirty day recovery period.