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additional toxicological information
Type of information:
experimental study
Adequacy of study:
supporting study
4 (not assignable)

Data source

Reference Type:
Effects of 2,2'-azobis (2-amidinopropane) hydrochloride in vivo and protection by vitamin E.
Dooley MM, Sano N, Kawashima H, Nakamura T
Bibliographic source:
Free Radical Biology & Medicine 9; 199-204

Materials and methods

Test guideline
no guideline followed
Principles of method if other than guideline:
To determine the uptake and lifetime of AAPH in rat plasma, four rats wete injected i.p. with 40 mg/kg AAPH administered in 2 mL/kg 0.9% saline solution. Blood (appr. 0.35 mL) was obtained in heparinized tubes from the tail veins of nonanesthetized rats prior to and 15 and 30 minutes and 1, 2, 3 and 5 hours after AAPH administration. Blood samples were centifuged at 12,000 rpm for 2 minutes and AAPH levels in plasma were determined by HPLC (ODP-50 reversed-phase column; AAPH was eluted with 20% (v/v) acetonitrile in 10 mM potassium phosphate buffer, pH 2.2, containing 5 mm sodium octane sulfonic acid. Peaks were detected at 210 nm.
To study AAPH-induced toxicity, rats were injected i.p. with 55 mg/kg AAPH in 2 mL/kg 0.9% saline solution. At various times (usually 18 hours) after administration, the animals were sacrificed (under sodium pentobarbital anesthesia). Blood was collected from the abdominal aorta into heparinized tubes, centrifuged at 12,000 rpm and plasma was stored on ice. Tissues to be analyzed were removed, weighed and homogenized in 1.15% potassium chloride solution. Tissue plasma lipid peroxidation was determined by measuring levels of thiobarbiruric acid-reactive substances (TBARS) by a spectrophotometric detection method in tissues or an HPLC detection method in plasma.
GLP compliance:

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2'-azobis[2-methylpropionamidine] dihydrochloride
EC Number:
EC Name:
2,2'-azobis[2-methylpropionamidine] dihydrochloride
Cas Number:
Molecular formula:
2-[(E)-2-(1-carbamimidoyl-1-methylethyl)diazen-1-yl]-2-methylpropanimidamide dihydrochloride
Details on test material:
- Name of test material (as cited in study report): AAPH

Results and discussion

Any other information on results incl. tables

Acute mortality (within 18 hours) in rats I.P exposed to AAPH was observed at and above 70 mg/kg bw (not indicated in method section of publication). AAPH doses between 50 and 60 mg/kg bw produced such signs of acute distress as tachypnea and crouching posture that appeared immediately after administration. In most rats these symptoms resolved slowly over several hours, although a few rats experienced increasing distress and died after several hours. For subsequent experiments a non-lethal dose of either 40 or 55 mg/kg bw was chosen.

HPLC chromatograms of rat plasma show a detection limit of 0.1 µg/mL plasma and peak areas were linear up to at least 1.4 mg AAPH applied. AAPH recoveries were 90 -92% over the entire concentration range examined.

Plasma levels of rats administered 40 mg/kg AAPH i.p. showed maximum AAPH levels in the earliest plasma sample collected (15 min), with a mean concentration of 25.11 ± 3.93 µg/mL and declined to 2.52 µg/mL at 3 hours after administration. The calculated half-life for AAPH in circulation is 29.9 minutes.

Examination of TBARS in plasma and tissues showed highest increase in the liver (2.5 -3 fold) 18 hours after administration. Significant increases were also observed in kidney and heart. Plasma levels of TBARS were more than 50% lower in the AAPH treated rats compared to controls. Liver TBARS rose within 1 hour of AAPH administration and reached a maximum after 8 hours, and remained fairly constant up to 24 hours.

The levels of total lipids, total cholesterol, triglycerides and phospholipids were all significantly lower in serum of AAPH treated rats compared to controls.

Applicant's summary and conclusion

Intraperitoneally administered AAPH at a dose level of 55 mg/kg bw in rats has a half-life of 29.9 minutes in plasma. Lipid peroxidation (measured by levels of thiobarbituric acid-reactive substances, TBARS) was observed in the liver, and to smaller extent in kidney and heart of AAPH treated rats. Compared to controls, lower levels of TBARS and lipid levels were observed in plasma of AAPH treated rats.