Pre-clinical Studies and Toxicology

All studies were performed at the Beijing Municipal Center for Hygiene and Epidemic Control in June of 1999. Four distinct studies were performed including: a maximal tolerated dose/LD50 experiment in mice, bone marrow micro-nucleus assay for mutagenicity, Ames test, and the mouse sperm cell deformity assay.

Summary Acute Oral Toxicity Study

The standard recommended dose in humans is 6-9 grams/day, which translates to 0.09-0.14 grams/kg assuming an average human weight of 65 kg (143 pounds). In the present study a combined total of 40 (20 male and 20 female) Kunming mice weighing between 18-22 grams were separated into 4 groups. The test substance (AcnEase^®) was administered orally at doses of 2.15, 4.64, 10.0 and 21.5 grams/kg body weight. This dose represents from approximately 24 to 240 times the standard human dose. Prior to dosing the animals were fasted overnight but allowed free access to water. After dosing the animals were allowed free access to food and water. The mice were observed for 2 weeks all signs and symptoms of toxicity and deaths were recorded.

Table 3

There were no overt signs of toxicity and no recorded deaths, therefore the LD50 determined for both male and females was in excess of 21.5 grams/kg which positions the product according to pharmacopoeial standards in the non-toxic category.

AMES Testing

The use of the Ames test is a standard method used to explore the mutagenic potential of test articles. The experiments are performed on the active ingredients directly and in a second test following incubation with mircrosomal enzymes. The drugs are incubated with the liver microsomes to mimic the effect of liver metabolism on the active ingredients to insure that metabolites of the drug do not possess mutagenic potential. The test is performed in 4 separate bacterial strains.

The results clearly demonstrate that AcnEase^® is not mutagenic in the Ames Assay when tested directly or following incubation with mircrosomal (S-9 preparation) enzymes. (The results of the test are on file)

Mouse Bone Marrow Micronucleus Assay

The purpose of these experiments is to determine if the test article (AcnEase^®) will induce chromosomal aberrations, which is an index of mutagenicity and carcinogenicity. 50 Kunming mice (25 males and 25 females) weighing between 35-30 grams were used in this study. The mice were divided into 5 groups: control, 2.5 grams/kg, 5.0 grams/kg and 10.0 grams/kg and the positive control Endoxan 40 mg/kg. The mice are orally administered the test articles twice in a 24 hour period. Six hours following the last dose polychromatic red blood cells are harvested and evaluated for chromosomal aberration.

Table 4

Results of Chromosomal Aberration Studies

The results clearly demonstrate that AcnEase^® treatment does not induce changes in bone marrow chromosomes supporting the position that the product is not likely to induce mutagenic are carcinogenic changes in cells.

Mice Sperm Deformity Assay

This assay is performed to determine if the test article (AcnEase^®) will induce aberrations in germ cell lines. The experiments were performed in 25 male Kunming mice 25-35 grams in weight. The mice were equally divided into 5 groups: control, 2.5 grams/kg, 5.0 grams/kg, 10.0 grams/kg of AcnEase^® and the positive control Endoxan 40 mg/kg, which was administered as an IP injection. The dosing regimen was once daily for 5 consecutive days. Thirty-five days after treatment the epididymis is isolated and 1000 sperm cells are examined for shape and deformity. The results demonstrate no statistically significant difference between the control group and any of the AcnEase^® tested groups. The results suggest that AcnEase^® does not adversely effect germ cell lines. (The results from this study are on file)