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A N I M P R O V E D M E T H O D F O R I N V I T R O T O X I C I T Y T E S T I N G

Submitted by

FRANK A. DE RENZIS AND JOSEPH J. ALEO

Departments of Periodontology and Pathology Temple University School of Dentistry

3223 North Broad Street Philadelphia, Pennsylvania 19140

I. I N T R O D U C T I O N

In the past, several methods (1-5) have been developed for the in vitro detection and assay of cyto- toxic agents on cultured cell monolayers. While a number of these methods have proven to be a relatively sensitive and reproducible, some are time-consuming, cumbersome, and at times incon- sistent and limited in their applicability. In spite of these difficulties, the procedures have proved extremely valuable for screening a variety of materials intended for use in medicine and other re- lated fields to determine their potential toxicity. One of the principal problems is the accommoda- tion of oddly shaped materials, and materials of low density. Not until the adoption of an agar diffusion method (6), which could be used for the test ing of solid as well as liquid extracts, was this problem alleviated. The technique described here offers an apparatus that modifies previously used in vitro screening methods to provide greater versatil i ty in accommodating objects of any shape or density. In addition, the accuracy of the results is increased by complete immobilization of the tes t sample.

Key words: toxicity; in vitro method; instrumentation; analysis.

II. M A T E R I A L S

Truchrome orthodontic wire, 0.036-inch, No. E-19 Rocky Mountain/Orthodontics 1

Round buccal tube, 0.036-inch, No. A-109' Erel-micro screw, smooth-housing, No. A-

5241 Erel-micro screw spring-loaded long piston, 8-

mm, No. A-5271 Erel-micro screw driver No. A-5281 Molar band material, 0.006-inch, No. B-109' How orthdontic band pinching pliers No. I-

1101 Orthodontic pliers, No. 1391 Or thodont ic welding unit, Rocky Mountain

Dial-A-Weld, No. J-506' Solder, 0.015-inch, regular silver, No. H-1191 Flux, Unitek Formula 6, No. 601-702 Unitek 2 Orthodontic gas-air soldering torch No. 603-

0142 Petri dish, Pyrex, 100- by 15-mm, No. 3483-

D31 Thomas 3

Rocky Mounta in /Or thodont ics , Denver CO. 2 Uni tek Corp., Monrovin CA. 3 A r t h u r H. Thomas , Philadelphia PA.

III. PROCEDURE

A. Retention band

1. Cut a section of molar band material (0.180 by 0.006 inch) slightly in excess of the circumference of a Petri dish.

2. With a How pliers, pinch the ends of the band material so as to create a t ight fit around the dish.

3. Weld the pinched ends of the band into a tab.

4. Bend this tab portion so that it rests flat against the band.

5. Make a series of welds and securely at- tach the tab portion to the band.

6. Seat the welded band over the Petri dish up to the lip, which serves as a stop (Fig. 1~]).

B. Buccal tube rests

1. Use a small file to make notches on the re- tention band at points selected for the location of buccal tube rests.

2. Weld the tube rests to the retention band so that they are horizontally orientated and opposite each other across the di- ameter of the Petri dish {Fig. 1 b,D.

775

C. Stabilizing bar

1. Select a piece of 0.036-inch orthodontic wire approximately 6 inches long.

2. Start at the end of the wire that fits into the mounted buccal tube rests and begin a series of bends that conform to the in- side surfaces of the Petri dish.

3. Leave a space of approximately 4 to 6 mm from the bot tom surface of the Petri dish. This space can vary, depending upon the thickness of the specimens to be tested.

4. Make the vertical arm bends of the sta- bilizing bar accurately so that they are of the same height (Fig. lc).

5. When seated over the dish, fit the sta- bilizing bar snugly and be careful to keep from shifting in order to prevent buckling of the bar.

D. Reinforcement arm

1. Cut a length of 0.036-inch orthodontic wire slightly larger than the diameter of the Petri dish.

2. With a No. 139 pliers, make a bend at the center of the wire so that the point of the band is in contact with the stabilizing bar and the ends of the reinforcement arm are in contact with the stabilizing bar jus t be- low the top edge of the Petri dish.

3. Spot weld adapted reinforcement arm at the center point.

4. Align the terminal ends of the reinforce- ment arms and solder to the stabilizing bar. It is important to flux all junctions before soldering {Fig. ld).

E. At tachment of micrel screws

1. Notch the stabilizing bar at points de- sired for placement of micrel screws.

2. Vary the number of micrel screws and the distance between each screw according to individual requirements.

a. Spot weld the micrel screws in position.

b. After proper alignment, perpendicular to the stabilizing bar, solder the micrel screws (Fig. le, g).

NOTE: See Fig. 2 for a complete assay assem- bly plate with test specimens in place.

IV. D I S C U S S I O N

The acceptable criteria for a satisfactory screening method are sensitivity, adaptabil- ity, speed and relatively low cost. While pre-

viously used methods were reported to be suc- cessful, each method had some inherent dis- advantage. The use of intramuscular im- plantation proved costly and time consum- ing. The use of the fluid medium technique {5) presented problems with specimens of low density or of odd shape or form, since the specimens had a tendency to float or to move about in the liquid medium. Also, this method was not practical for the test ing of liquids. The disadvantage of the agar diffusion method (6} was the thickness of the agar over- lay, which could absorb a portion of the toxic component and thereby reduce the concentra- tion reaching the cell monolayer. Reducing the thickness of the agar overlay would rem- edy this but, in turn, would reduce the poten- tial longevity of the underlying cell mono- layer.

The use of the plate assembly described in this paper makes it possible to stabilize speci- mens of any shape or density. The cells can be fed, if necessary, without any disturbance of the specimen. Extracts or solution can be tested by the use of filter-paper discs satur- ated with the test material. By placing the specimens to be tested directly onto the cell monolayer, a more sensitive assay is possible. The entire assembly is easily constructed and low in cost, can be autoclaved and may be used repeatedly.

V. R E F E R E N C E S

1. Eagle, H., and G. E. Foley. 1956. Cytotoxic action of carcinolytic agents in tissue cul- ture. Am. J. Med. 21: 739-749.

2. Miyamura, S. 1956. A determination method for anticancer action of antibiotics by the agar plate diffusion technique. Antibiot. Chemother. 6: 280-282.

3. Brewer, J. H., and H. H. Bryand. 1960. The toxicity and safety testing of disposable medical and pharmaceutical materials. J. Am. Pharm. Assoc. 49: 652-656.

4. Renis, H .E . , H .G. Johnson, and B .K. Bjuyan. 1962. A collagen plate assay for cytotoxic agents. I. Methods. Cancer Res. 22: 1126-1130.

5. Rosenbluth, S. A., G. R. Weddingdon, W. L. Guess, and J. Autian. 1965. Tissue culture method for screening toxicity of plastic materials to be used in medical practice. J. Pharm. Sci. 54: 156-159.

6. Guess, W. L., S. A. Rosenbluth, B. Schmidt, and J. Autian. 1965. Agar diffusion method for toxicity screening of plastics on cultured cell monolayers. J. Pharm. Sci. 54: 1545-1547.

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