SAFETY TESTING OF COSMETICS 137 age levels. The rats are observed for gross signs of systemic toxicity, pharma- cological effects, and mortality at frequent intervals during the day of dosage, and at least once daily thereafter for 14 days. Gross autopsies are performed on any animals xvhieh die. Acute Eye Irritation Six albino rabbits are used in these tests. One-tenth milliliter of a liquid test material, or 100 mg of solids or pastes, is placed in one eye of each of the rabbits. The opposite eye is untreated and serves as a control. Readings for eye irritation are made at 24, 48, and 72 hours following application. The scor- ing scale for recording eye irritation is that of Draize (2). Primary Skin Irritation and Corrosivity The test material is applied under a 1-in. 2 gauze patch to both an intact and an abraded skin area on each of six albino rabbits, whose trunks have been clipped free of fur. Four patches can be applied, allowing for two sam- ples to be tested on each animal. Acute Derreal Toxicity This study uses four groups of four albino rabbits. A single derreal appli- cation of the test material is made to each group at graduated dosage levels. The skin of two rabbits per group is abraded the skin of the two remaining rabbits per group remains intact. The test material is applied under a rubber dental dam and remains in contact with the skin for 24 hours while the ani- mals are restrained in stocks. Acute Inhalation Toxicity Ten male albino rats are exposed in an inhalation chamber for one hour to an atmospheric concentration .of 20,000 ppm of gas .or vapor or 200 mg/1. of mist or dust. The rats are observed during the exposure period and daily for 14 days for gross signs of systemic toxicity. Gross autopsies are performed on rats which die. Standard criteria are used to evaluate the results of 'these various animal studies as shown in Table I. Subacute Derreal (21-Day) Toxicity This study evaluates the percutaneous absorption and irritative potential of the test material following subacute derreal application to rabbits. The test is not specified by the regulation but is frequently carried out. Eighteen young adult albino rabbits are divided randomly into three groups of six rabbits each. Group 1 is the control group and receives repeated skin applications of dis- tilled water. Group 2 receives repeated skin applications of the test material

JOURNAL OF TIlE SOCIETY OF COSMETIC CHEMISTS Table I Evaluation of Toxicity in Animal Studies Route of Administration Highly Toxic Toxic Nontoxic Oral Derreal Inhalation LD5o of 50 mg/kg or less LD5o of 200 mg/kg or less LD5o of 200 ppm of gas or vapor (2 rag/1. for mist or dust) or less during one- hour exposure LD,o 50 mg/kg but LD•,o g/kg not 5 g/kg LD,o 200 mg/kg but LD•o 2 g/kg not 2 g/kg LD5o 200 ppm of gas LD,o 20,000 ppm or vapor (2 mg/]. for of gas or vapor mist or dust) but not (200 rag/1. of more than 20,000 ppm mist or dust) (200 rag/1. of mist or dust) at a low dosage level. Group 3 receives repeated skin applications of the test material at a high dosage level ( 10 times that of Group 2). Three rabbits in each group are abraded at the beginning of the first, sec- ond, and third weeks of the study. The skin of the remaining rabbits is left intact. The control and test materials are applied once daily on a 5-day per week basis over a 3-week period, for a total of 15 applications. Each daily ex- posure period is for 6 to 8 hours. Following the 15 applications, the rabbits are observed for 2 weeks, then sacrificed, and gross necropsies are performed. During the study each animal is weighed weekly and observed daily for gross signs of derreal irritation and systemic toxicity. A complete blood count, hemoglobin determination, and urinalysis are performed initially and at ter- mination of the study. At necropsy, representative tissues from each control and test animal are preserved in 10% formaldehyde. Microscopic examination of tissues is made on each control and high-level rabbit, including the skin, heart, liver, kidney, spleen, adrenal, stomach, small intestine, gonad, and bone marrow. From the low-level group, sections of skin, liver, and kidney are examined. All remaining tissues from all animals are held for the possibility of further micrscopie examinations. An evaluation which frequently is carried out in guinea pigs is for detection of allergenic potential of test materials. This procedure should be applied solely in the evaluation of allergenic potential of proposed product compo- nents. The evaluation of proposed market formulations is usually restricted to tests on human panelists. In reviewing the applications of the guinea pig test for sensitization, the general opinion is that the production of positive reactions in guinea pigs should exclude the material from further consideration for use in preparations which will be topically applied to the skin, or it should indicate caution in subsequent tests on humans which may follow in order to determine concen-