Registration Dossier

Administrative data

Endpoint:
neurotoxicity
Remarks:
acute
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Cross-reference
Reason / purpose:
reference to same study

Data source

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
The cerebral hemispheres of intraperitoneally exposed rats were taken at autopsy and were analysed for RNA, Glutathione (CAS 70-18-8), Acetylcholine esterase (CAS 9000-81-1) and Succinate dehydrogenase (CAS 9002-02-2) activities.
GLP compliance:
no
Limit test:
yes

Test animals

Species:
rat
Strain:
Wistar
Sex:
male

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
olive oil
Remarks:
60 mg test item / mL olive oil
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
1, 3 or 7 days
Doses / concentrations
Remarks:
Doses / Concentrations:
100 mg test item / kg body weight
Basis:
other: actually injected (i.p.)
No. of animals per sex per dose:
5 (in each of the three treatments and the control, altogether 20 animals)
Control animals:
yes, concurrent vehicle

Examinations

Statistics:
The results were evaluated using the Student's T-test.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No mortality and no clinical effects were reported
Mortality:
no mortality observed
Description (incidence):
No mortality and no clinical effects were reported
Body weight and weight changes:
not examined
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
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Cerebral Glutathione concentration below control level after 1 day but returned to control value after 3 and 7 days postinjection; Brain Acetylcholine esterase activity marginally decreased after 1 day only, while RNA and Succinate dehydrogenase unchanged
Behaviour (functional findings):
not examined
Gross pathological findings:
not examined
Neuropathological findings:
no effects observed
Description (incidence and severity):
No gross-pathological effects in the investigated brain hemispheres reported
Other effects:
not examined

Any other information on results incl. tables

Table 1: Neurochemical effects of intraperitoneal Ethylhexyl nitrate

Time [d]

RNA [γ/mg protein]]

Glutathione [ng/mg protein]

Acetylcholine esterase [nmol/(min mg protein)]

Succinate dehydrogenase [nmol/(min mg protein)]

1

22.3±1.9

551±24 (c)

190±8 (b)

3.2±0.14

3

21.1±1.7

593±46

187±13 (a)

3.3±0.24

7

21.9±3.0

624±24

185±22

3.2±0.31

Control

24.0±4.6

610±23

203±8

3.1±0.22

Each figure is the mean of 5 test animals ± SD

SD = Standard Deviation

(a: p<0.05

(b): p<0.01

(c): p<0.001

The nitroester dose caused a decrease in Glutathione concentration and an initial decrease in acetylcholine concentration. However, there was no significant effects on RNA and mitochondrial Succinate dehydrogenate levels. This decrease is compatible with the Glutathione dependent deesterification of Glyceryl trinitrate found in vitro (Heppel & Hilmoe 1950) and in vivo (Zitting & Slavolainen 1982).

  • Heppel LA, Hilmoe RJ (1950). Metabolism of inorganic nitrite and nitrate esters. II. The enzymatic reduction of nitroglycerin and erythritol tetranitrate by glutathione. The Journal of Biological Chemistry 183:129-38. URL http://www.jbc.org/content/183/1/129.full.pdf
  • Zitting A, Savolainen H (1982). Effects of nitroglycerin and ethylene glycol dinitrate mixture (blasting oil) on rat brain, liver and kidney. PMID 6812185 Res Commun Chem Pathol Pharmacol 37(1):113-21.

Applicant's summary and conclusion

Executive summary:

There was no major structural damage caused by the high exposure to ethylhexyl nitrate becasue the effects were only temporary and the enzyme activity is dependent on the lipid structure of the excitable membrance, which was not damaged. The authours could not explain the decrease in Acetylcholine esterate, but it is known from the literature that tetra N-alkylammonium ions interfere with the esterase activity.