V. ESTIM/VTIGN 511 



adequacy of the test dose. The assay period is thereby shortened, and there 

 is no temptation to use the animal for further and possibly unreliable as- 

 says. 



There is some question as to the time and manner of administering the 

 test dose, whether on the fourth, fifth, and sixth days,^^ or on each of the 

 first ten days,^° after conception; the latter should be advantageous if more 

 efficient use can be made of a smaller dose.^^ The test substance is ordinarily 

 administered bj^ mixing it with a small portion of the diet under conditions 

 that assure its consumption or by giving it, dissolved in a suitable oil 

 (olive), with a medicine dropper. 



Enough animals must be used (perhaps ten) on each of several levels of 

 ingestion (three or four) to permit statistical treatment of the results which 

 relate dosage to litter efficiency either on a curve^^ or more simply by the 

 method of probits.^^ Litter efficiency is suitably expressed in terms of mean 

 fertilit\^ dose, which is the least amount of tested substance on which one- 

 half of the animals give a positive response as defined above. A series of 

 animals on pure tocopherol or an ester of it should accompany the several 

 series of the unknown substance under test. 



The results of bioassays made by this standardized procedure are more 

 reliable than earlier results, many of which were disappointing in their 

 wide divergence; thus, the mean fertility dose for synthetic racemic dl-a- 

 tocopherol acetate was 0.56 to 1.71 mg.^* The MFD of natural a-tocopherol 

 (and its succinic acid ester) given by mouth is now 0.75 mg.^" The "unit" 

 for vitamin E as originally proposed^^ was 1 mg. of tocopherol acetate in 

 olive oil, an amount greater than the presently accepted MFD for this sub- 

 stance. 



Other criteria for the basis of a bioassay have included increase in ma- 

 ternal body weight during gestation, ^^ the number of offspring delivered 

 at term, whether living or dead,^® or the percentage of placental implants 

 resulting in birth of living offspring. ^^ The disappearance of symptoms of 

 exudative diathesis in chicks,®^ the reduction of creatinuria in rabbits suf- 

 fering from muscular dystrophy, the prevention of hemolysis due to an 



" M. Joffe and P. L. Harris, J. Am. Chem. Soc. 65, 925 (1943). 



6" P. L. Harris, J. L. Jensen, M. Joffe, and K. E. Mason, J. Biol. Chem. 156, 491 (1944). 



" C. A. Cabell and N. R. Ellis, /. Nutrition 23, 633 (1944). 



82 A. L. Bacharach, Biochem. J. 32, 2017 (1938). 



" K. E. Mason and P. L. Harris, Biol. Symposia 12, 459 (1947). 



"E. M. Hume, Nature 148, 472 (1941); Quart. Bull. Health Organisation League 



Nations 9, 4.36 (1940-1941). 

 " H. Gottlieb, F. W. Quackenbush, and H. Steenbock, /. Nutrition 25, 433 (1943). 

 "A. L. Bacharach and E. AUchorne, Biochem. J. 32, 1298 (1938). 

 "L. S. Palmer, Ind. Eng. Chem. Anal. Ed. 9, 427 (1937). 

 ^' H. Dam, J. Glavind, I. Prange, and J. Ottesen, Kgl. Danske Videnskab. Selskab. 



Biol. Medd. 16, No. 7 (1941). 



