Registration Dossier

Administrative data

Endpoint:
mechanistic studies
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
October 14, 1988 to October 27, 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Non-GLP experiment performed in a contract research organization, reported in sufficient detail including raw data, but one page partly missing in the PDF available

Data source

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Antihypertensive assay; Aortic intubated rats were intra-arterially dosed with equimolar 2-Ethylhexyl Nitrate, Nitroglycerin (CAS 55-63-0) or Propylene glycol dinitrate (CAS 6423-43-4). The test animals were cannulated for systolic and diastolic blood pressure and heart rate monitoring according to the method published by Weeks & Jones (1960 Proc. Soc. Exptl. Biol. Med. 104:646).
GLP compliance:
no
Remarks:
No GLP quality assurance statement is made
Type of method:
in vivo
Endpoint addressed:
other: Effects on heart rate and blood pressure

Test animals

Species:
rat
Strain:
other: Spontaneously Hypertensive (SH) rats
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Wilmington, Massachusetts, U.S.A.
- Age at study initiation: 12 -14 weeks
- Housing: Individually or in groups, according to sex, in stainless steel ½" wire mesh cages, size in accordance with “Guide for the Care and Use of Laboratory Animals”
- Diet: Ad libitum
- Water: Ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3 °C
- Humidity (%): 40-70 %
- Photoperiod (hrs dark / hrs light): 12 /12

Administration / exposure

Route of administration:
other: intra-arterially, volume of administration 2 mL/kg bw
Vehicle:
ethanol
Remarks:
50 %
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
1 day
Frequency of treatment:
Once
Post exposure period:
48 h
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 0.8, 4 and 8 µmol/kg bw
Basis:
other: actual injected
No. of animals per sex per dose:
6
Control animals:
yes, concurrent vehicle

Examinations

Examinations:
Systolic and diastolic blood pressure and the heart rate were monitored
Positive control:
Among the 3 test items Nitroglycerin (CAS 55-63-0) was known to produce significant effects and served thus as positive control.

Results and discussion

Details on results:
The equimolar doses produced the following effects (in brackets the statistically significant levels are given, p<0.05) in the comparably tested Organonitrates:

2-Ethylhexyl Nitrate (the registered substance, branched Organomononitrate)
-Systolic blood pressure: Decrease (8 µmol/kg bw), antagonisation of the ethanol effect
-Diastolic blood pressure: Decrease (8 µmol/kg bw), antagonisation of the ethanol effect
-Heart rate: Increase (no statistical significance), by tendency synergism with the ethanol effect

Propylene glycol dinitrate (CAS 6423-43-4, branched Organodinitrate)
-Systolic blood pressure: Decrease (0.8 µmol/kg), antagonisation of the ethanol effect
-Diastolic blood pressure: Decrease (no statistical significance), antagonisation of the ethanol effect
-Heart rate: Increase (4 and 8 µmol/kg), by tendency synergism with the ethanol effect

Nitroglycerin (CAS 55-63-0, positive control, unbranched Organotrinitrate)
-Systolic blood pressure: Decrease at all doses; 0.8, 4 and 8 µmol/kg bw, antagonisation of the ethanol effect
-Diastolic blood pressure: Decrease at all doses; 0.8, 4 and 8 µmol/kg bw, antagonisation of the ethanol effect
-Heart rate: Increase at all doses; 0.8, 4 and 8 µmol/kg bw, by tendency synergism with the ethanol effect

Vehicle control (all effects statistically significant, p<0.05)
-Systolic blood pressure: Increase
-Diastolic blood pressure: Increase
-Heart rate: Increase

Mortality within the 48 posttreatment observation period was as follows:
2-Ethylhexyl Nitrate: 7 (3 at the low dose, 2 at the mid dose and 2 at the high dose)
Propylene glycol dinitrate: 3 (1 at each dose level)
Nitroglycerin: 4 (1 at the low dose, 1 at the mid dose and 2 at the high dose)
Vehicle control: 2

Applicant's summary and conclusion

Conclusions:
Organonitrates act similarly on heart rate and blood pressure. While the trinitrate had a stronger effect, demonstrated by the fact that only with the positive control statistical significance was reached in all tested levels, no difference in the strength of effects is visible between the di- and the mononitrate. Considering the equimolar doses, the potential nitrogen monoxide release (known to be the pharmacological principle of nitroglycerin) was three times higher than the one of 2-Ethylhexyl nitrate or PGDN. This suggests that GDN will serve as a worst case scenario for 2-EHN, and PGDN will provide satisfactory read across data for 2-EHN.
Executive summary:

The cardiovascular effects of equimolar doses of organonitrates including the registered substance 2-Ethylhexyl nitrate (CAS 27247-96-7) as mononitrate, Propylene glycol dinitrate (CAS 6423-43-4) and Nitroglycerin (CAS 55-63-0, positive control) as trinitrate were comparatively investigated employing an antihypertensive assay.

Using a volume of 2 mL/kg bw, ten groups of 6 male Spontaneously Hypertensive (SH) rats received one intra-arterially treatment with 0.8, 4 or 8 µmol/kg bw of each test item (50 % ethanol solution) or the vehicle control. Intentionally the vehicle control increased all parameters monitored, which were Systolic blood pressure, Diastolic blood pressure and the Heart rate.

All tested organonitrates antagonize the increase of systolic and diastolic blood pressure caused by the vehicle and have little effect on the heart rate seemingly causing a slightly higher increase as the vehicle alone (weak synergism).

The comparison of tri-, di-, and mononitrate substances reveals by tendency that the organonitrates act similarly on heart rate and blood pressure, but the trinitrate clearly has a stronger effect, demonstrated by the fact that only with this agent statistical significance was reached in all tested levels. No difference in the strength of effects is visible between the di- and the mononitrate, but the statistical power is low and only a few treatments were statistically different to the pre-exposure measurements.

In conclusion organonitrates act similarly on heart rate and blood pressure in the experimental setting used. While the trinitrate had a stronger effect, demonstrated by the fact that only the positive control statistical significance was reached in all tested levels, no difference in the strength of effects is visible between the di- and the mononitrate. Considering the equimolar doses, the potential Nitrogen monoxide release (known to be the pharmacological principle of Nitroglycerin) was three times higher than the one of 2-Ethylhexyl nitrate.This suggests that GDN will serve as a worst case scenario for 2-EHN, and PGDN will provide satisfactory read across data for 2-EHN.