very 2 centimeters or 3 centimeters ongitudinally over the area of exposure. The abrasions are sufficiently deep to enetrate the stratum corneum (horny ayer of the epidermis), but not to disurb the derma-that is, not to obtain leeding.

(c) Procedures for testing. The sleeve s slipped onto the animal, which is then placed in a comfortable but immobilized position in a multiple animal holder. Selected doses of liquids and solutions are ntroduced under the sleeve. If there is light leakage from the sleeve, which may occur during the first few hours of exDosure, it is collected and reapplied. Dosage levels are adjusted in subsequent exposures (if necessary) to enable a calculation of a dose that would be fatal o 50 percent of the animals. This can be determined from mortality ratios obCained at various doses employed. At the end of 24 hours the sleeves or screens are removed, the volume of unabsorbed material, if any, is measured, and the skin reactions are noted. The subjects are cleaned by thorough wiping, observed for gross symptoms of poisoning, and then observed for 2 weeks.

§ 191.11 Method of testing primary irritant substances.

Primary irritation to the skin is measured by a patch-test technique on the Fabraded and intact skin of the albino rabbit, clipped free of hair. A minimum of six subjects are used in abraded and intact skin tests. Introduce under a square patch such as surgical gauze measuring 1 inch x 1 inch, two single layers thick, 0.5 milliliter (in case of liquids) or 0.5 gram (in the case of solids and semisolids) of the test substance. Dissolve solids in an appropriate solvent and apply the solution as for liquids. The animals are immobilized with patches secured in place by adhesive tape. The entire trunk of the animal is then wrapped with an impervious material such as rubberized cloth for the 24-hour period of exposure. This material aids in maintaining the test patches in position and retards the evaporation of volatile substances. After 24 hours of exposure, the patches es are removed and the resulting reactions are evaluated on the basis of the designated values in the following table:

1 The "value" recorded for each reading is the average value of the six or more animals subject to the test.

Readings are again made at the end of a total of 72 hours (48 hours after the first reading). An equal number of exposures are made on areas of skin that have been previously abraded. The abrasions are minor incisions through the stratum corneum, but not sufficiently deep to disturb the derma or to produce bleeding. Evaluate the reactions of the abraded skin at 24 hours and 72 hours, as described in this paragraph. Add the values for erythema and Eschar formation at 24 hours and at 72 hours for intact skin to the values on abraded skin at 24 hours and at 72 hours (four values). Similarly, add the values for edema formation at 24 hours and at 72 hours for intact and abraded skin (four values). The total of the eight values is divided by four to give the primary irritation score. Example:

(a) (1) Six albino rabbits are used for each test substance. Animal facilities for such procedures shall be so designed and maintained as to exclude sawdust, wood chips, or other extraneous materials that might produce eye irritation. Both eyes of each animal in the test group shall be examined before testing, and only those animals without eye defects or irritation shall be used. The animal is held firmly but gently until quiet. The test material is placed in one eye of each animal by gently pulling the lower lid away from the eyeball to form a cup into which the test substance is dropped. The lids are then gently held together for one second and the animal is released. The other eye, remaining untreated, serves as a control. For testing liquids, 0.1 milliliter is used. For solids or pastes, 100 milligrams of the test substance is used, except that for substances in flake, granule, powder, or other particulate form the amount that has a volume of 0.1 milliliter (after compacting as much as possible without crushing or altering the individual particles, such as by tapping the measuring container) shall be used whenever this volume weighs less than 100 milligrams. In such a case, the weight of the 0.1 milliliter test dose should be recorded. The eyes are not washed following instillation of test material except as noted below.

(2) The eyes are examined and the grade of ocular reaction is recorded at 24, 48, and 72 hours. Reading of reactions is facilitated by use of a binocular loupe, hand slit-lamp, or other expert means. After the recording of observations at 24 hours, any or all eyes may be further examined after applying fluorescein. For this optional test, one drop of fluorescein sodium ophthalmic solution U.S.P. or equivalent is dropped directly on the cornea. After flushing out the excess fluorescein with sodium chloride solution U.S.P. or equivalent, injured areas of the cornea appear yellow; this is best visualized in a darkened room under ultraviolet illumination. Any or all eyes may be washed with sodium chloride solution U.S.P. or equivalent after the 24hour reading.

(b) (1) An animal shall be considered as exhibiting a positive reaction if the

test substance produces at any of the readings ulceration of the cornea (other than a fine stippling), or opacity of the cornea (other than a slight dulling of the normal luster), or inflammation of the iris (other than a slight deepening of the folds (or rugae) or a slight cir cumcorneal injection of the blood ves sels), or if such substance produces in the conjunctivae (excluding the corne and iris) an obvious swelling with par tial eversion of the lids or a diffuse crimson-red with individual vessels no easily discernible.

(2) The test shall be considered positive if four or more of the animals in the f test group exhibit a positive reaction. I only one animal exhibits a positive reaction, the test shall be regarded as negative. If two or three animals exhibit a positive reaction, the test is repeated using a different group of six animals. The second test shall be considered positive if three or more of the animals exhibit a positive reaction. I only one or two animals in the second test exhibit a positive reaction, the tes shall be repeated with a different group of six animals. Should a third test be needed, the substance will be regarded as an irritant if any animal exhibits a positive response.

(c) To assist testing laboratories and other interested persons in interpreting the results obtained when a substance is tested in accordance with the method described in paragraph (a) of this section, an "Illustrated Guide for Grading Eye Irritation by Hazardous Substances" will be sold by the Superintendent of Documents, Government Printing Office, Washington, D.C. The guide will contain color plates depicting responses of varying intensity to specific test solutions. The grade of response and the, substance used to produce the response will be indicated.

[29 F.R. 13009, Sept. 17, 1964]

§ 191.13 Tentative method of test for flashpoint of volatile flammable ma terials by Tagliabue open-cup appa

SCOPE

ratus.1

12

1. (a) This method describes a test procedure for the determination of open-cup

1 The Food and Drug Administration has obtained permission from the American So

lashpoints of volatile flammable materials aving flashpoints below 175° F.

(b) This method, when applied to paints nd resin solutions which tend to skin over r which are very viscous, gives less reprotucible results than when applied to olvents.

OUTLINE OF METHOD

2. The sample is placed in the cup of a Tag Open Tester, and heated at a slow but constant rate. A small test flame is passed it a uniform rate across the cup at specified ntervals. The flashpoint is taken as the owest temperature at which application of che test flame causes the vapor at the surface of the liquid to flash, that is, ignite but not continue to burn.

APPARATUS

3. The Tag open-cup tester is illustrated in Fig. 1. It consists of the following parts,

which must conform to the dimensions shown, and have the additional characteristics as noted:

(a) Copper bath, preferably equipped with a constant level overflow so placed as to maintain the bath liquid level 1⁄2 inch below the rim of the glass cup.

(b) Thermometer holder. Support firmly with ringstand and clamp.

(c) Thermometer. For flashpoints above 40° F., use the ASTM Tag Closed Tester Thermometer, range of +20 to +230° F., in 1° F. divisions, and conforming to thermometer 9F. of ASTM Standard E 1. For flashpoints from 20° F. to 40° F., use ASTM Tag Closed Tester, Low Range, Thermometer 57F. For flashpoints below 20° F., use ASTM Thermometer 33F. The original Tag Open-Cup (Paper Scale) Thermometer will be a permissible alternate until January 1, 1962. It is calibrated to -20° F.

(d) Glass test cup. Glass test cup (Fig. 2), of molded clear glass, annealed, heat-resistant, and free from surface defects.

(e) Leveling device. Leveling device or guide, for proper adjustment of the liquid

ciety for Testing Materials, Philadelphia, Pa., to reprint this method in these regulations. The text has been slightly modified, for practical reasons.

' ASTM Designation: D 1310-59T, issued 1954; revised 1955, 1956, 1959. This tentative method has been approved by the sponsoring committee and accepted by the American Society for Testing Materials in accordance with established procedures, for use pending adoption as standard. Suggestions for revisions should be addressed to the Society at 1916 Race St., Philadelphia, Pa.

level in the cup (Fig. 3). This shall be made of No. 18-gage polished aluminum, with a projection for adjusting the liquid level when the sample is added to exactly %-inch below the level of the edge or rim of the cup.

(f) "Micro," or small gas burner of suitable dimensions for heating the bath. A screw clamp may be used to help regulate the gas. A small electric heater may be used.

(g) Ignition taper, which is a small straight, blow-pipe type gas burner. The test flame torch prescribed in the method of test for flash and fire points by Cleveland Open Cup (ASTM designation: D 92) is satisfactory.

(h) Alternative methods for maintaining the ignition taper in a fixed horizontal plane above the liquid may be used, as follows:

(1) Guide wire, 2-inch in diameter and 31⁄2 inches in length, with a right-angle bend 1⁄2-inch from each end. This wire is placed snugly in holes drilled in the rim of the bath, so that the guide wire is %-inch from the center of the cup and resting on the rim of the cup.

(2) Swivel-type taper holder, such as is used in ASTM METHOD D 92. The height and position of the taper are fixed by adjusting the holder on a suitable ringstand support adjacent to the flash cup.

(1) Draft shield, consisting of two rectangular sheets of noncombustible material, 24 inches x 28 inches, are fastened together along the 28-inch side, preferably by hinges. A triangular sheet, 24 inches x 24 inches x 34 inches is fastened by hinges to one of the lateral sheets (to form a top when shield is open). The interior of the draft shield shall be painted a flat black.

PROCEDURE

4. (a) Place the tester on a solid table free of vibration, in a location free of perceptible draft, and in a dim light.

(b) Run water, brine, or water-glycol solution into the bath to a predetermined level, which will fill the bath to %-inch below the top when the cup is in place. An overflow is permissible for water-level control.

(c) Firmly support the thermometer vertically halfway between the center and edge of the cup on a diameter at right angles to the guide wire, or on a diameter passing through the center of the cup and the pivot of the taper. Place so that the bottom of the bulb is 1⁄4-inch from the inner bottom surface of the cup. If the old Tagliabue thermometer is used, immerse to well cover the mercury bulb, but not the wide body of the thermometer.

(d) Fill the glass cup with the sample liquid to a depth just -inch below the edge, as determined by the leveling device.

(e) Place the guide wire or swivel device in position, and set the draft shield around