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Control Series — Published Quarterly — May Issue, 1937 — No. 162
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Commercial Fertilizers, Commercial Feeds

AND

Agricultural Liming Materials

A Summary of Vitamin D Assay Work
in Maryland

STATE INSPECTION SERVICE
Controlled by
UNIVERSITY OF MARYLAND
College Park, Md.

SSS oo
Entered at College Park (Md.) Post Office as Second Class Matter.

A SUMMARY OF VITAMIN D ASSAY WORK IN MARYLAND

As a result of the general recognition of the importance of an adequate
supply of vitamin D in poultry rations, the market for anti-rachitic supplements
has undergone great expansion during the past several years. The majority of
commercial poultry mashes registered by the Maryland Feed Inspection Service
now contain some ingredient included as a vitamin D supplement. Also large
quantities of cod liver oil and other vitamin D carriers are retailed to livestock
and poultry feeders who mix their own rations.

As early as 1930, Feed Control Officials appreciated the trend in the use
of vitamin D supplements and foresaw the need for testing such products as a
routine regulatory function.

At that time, the potency of vitamin D carriers was universally determined
by means of the rat assay, a technic which had been highly developed. Most of
the vitamin D supplements going into the feed industry were being used in
poultry rations, however, and it was felt that a method of assay involving chick
feeding would be more serviceable for feed control work. The demonstration that
rats and chicks do not react in the same relative degree to vitamin D from various
sources further amplified the advisability of a chick assay test for products
designed for poultry feeding purposes.

As the result of experimental work in the Minnesota and Wisconsin feed
control laboratories a procedure was developed which seemed promising. The
method was tried out in several laboratories and found to give acceptable com-
parative results. With minor alterations the procedure was adopted as a
tentative method by the Association of Official Agricultural Chemists in 1933.

The test, which became known as the A. O. A. C. method, is based on the
percentage of ash produced in the tibiae of chicks fed for a four weeks period on
a specific basal rachitic ration to which is added varying amounts of the product
under assay. Chicks fed the basal ration alone become severely rachitic within
four weeks, and show tibiae ash contents of from 30-34%. Chicks fed the basal
ration supplemented with an adequate amount of vitamin D show tibiae ash con-
tents of 44-48%. With insufficient vitamin D supplement the ash of the tibiae
will fall in the range between 34% and 44% depending upon the severity of the
deficiency.

A comparison of the potency of various oils is possible with the method
by determining the smallest percentage of each which is just sufficient to produce
a maximum ash content. In illustration, if it is found that the basal ration must
be supplemented with %% of oil A, 4% of oil B, and 4%% of oil C, in order to
produce tibiae ash averages of 44% in chicks fed these supplemented rations,
then oil A is approximately twice as potent in vitamin D as oil B, and oil B
in turn is approximately twice as potent as oil C.

(A detailed description of the A. O. A. C. method as revised in 1936 is given
on page 4 of this paper.)

In 1932, subsequent to a ruling by the Attorney General that products sold as
vitamin carriers for poultry and livestock feeding were covered by the Maryland
Feed Law, the Maryland Feed Inspection Service began the collection and assay
of samples of vitamin D supplements being merchandised in Maryland. The
procedure at first followed was to supplement the basal ration of the A. O. A. C.
method with %, %, and %*%% of the respective products in the case of cod liver
oils, and higher percentages of lower potency materials such as cod liver meal.
The response in terms of bone ash production allowed a comparison of the
potency of the various samples within the limits of the percentage ranges chosen.
The results of these tests were published in Inspection Service bulletins.

Beginning January 1, 1933, all brands of vitamin D carriers were required
to be registered with the Inspection Service, and certain labeling requirements
were put into effect. The following year, in accord with recommendations of the
Association of American Feed Control Officials, the Maryland Inspection Service

3

required also that the registration and label should carry a statement of guar-
anteed minimum potency by the A. O. A. C. method of assay. This potency was
expressed as the percentage of the product which when added to the basal ration
of the A. O. A. C. method would produce a minimum bone ash of 44%.

Samples were collected twice yearly when possible and tests made to determine
whether the products were of the potency guaranteed. In reporting the test
results, if the ash percentage found was 44% or more at the guaranteed level
the product was reported as “Satisfactory.” If a deficiency was found the test
was reported as “Unsatisfactory.”

This same procedure was followed in 1935 and 1936 except that the term
“Guarantee Sustained” was substituted for “Satisfactory,” and “Guarantee Not
Sustained” was used in place of “Unsatisfactory” in reporting test results. Also
in cases of deficiency the actual ash percentage obtained was shown.

During the years from the introduction of the chick test up to 1936 work
was being conducted in several laboratories, notably in Minnesota and Wisconsin,
in an effort to refine and improve the utility of the A. O. A. C. method. One of
the most significant lines of study, involved the attempt to devise a means of
expressing potency as evaluated by the A. O. A. C. method in terms of units
of vitamin D. Facilitated by the availability of the accurately assayed U. S.
Pharmacopoeia “Reference Oil,” it was found possible to measure the response
of chicks to rations carrying definite unitages of vitamin D. By comparing the
bone ash of chicks fed rations of known vitamin D content from the reference oil
with the ash percentages produced by definite percentage levels of test oils, it
was found possible to calculate the potency of the latter in terms of units. The
potency of the U. S. Pharmacopoeia “Reference Oil” is expressed in terms of
“U.S. P. units” which are evaluated on the basis of a rigidly standardized rat
test. Quite obviously, the results of the chick test could not be expressed as
“U.S. P. units.” An A. O. A. C. chick unit was thus devised and is described
adequately in the following resolution adopted at the 1936 meeting of the
Association of American Feed Control Officials: ‘Resolved that any material
sold primarily as a vitamin D supplement for poultry shall be labeled with a
minimum guarantee of vitamin D potency expressed as Association of Official
Agricultural Chemists’ chick units of vitamin D per gram, determined according
to the tentative procedure adopted by the Association of Official Agricultural
Chemists and assuming that one U. S. P. unit of vitamin D from the U. S.
Pharmacopoeia “Reference Oil” equals one Association of Agricultural Chemists’
chick unit.” The A. O. A. C. method was revised in 1936 so as to provide for
evaluation in terms of units. At the same time the four weeks feeding period
previously specified was reduced to a three-week period.

Following the resolution quoted above the Maryland Feed Inspection Service
adopted the chick unit guarantee as registration and labeling requirements for
1937.

Immediately following is a detailed description of the recently revised
A. O. A. C. method.

Vitamin D Assay By Preventive Biological Test

(Applicable to fish and fish liver oils and their extracts, and to
materials used for supplementing the vitamin D content of feeds. Not
applicable to irradiated ergosterol products or to irradiated yeast unless
recommended for poultry.)

Basal Rachitic Ration

Parts
Groundwy.ellow, connie seat see eles te ee 59
Pure wheat flour middlings.....___ 25
Crude domestic acid precipitated casein SZ
Calcium carbonate (precipitated) _____ ks 1
Calcium phosphate (precipitated) _____ Jn ol
Todized salt (0.02% potassium iodide)... =. 3
Non-irradiated yeast (7% minimum nitrogen)... 1

4

Procedure

Place groups of 10 or more 1-day-old White Leghorn chickens in
screen-bottomed biological cages or battery brooders out of direct sunlight.
(Red electric light bulbs are satisfactory as a source of heat for the
cages.) Reserve one group for negative control purposes, one or more
groups for positive control purposes, and one or more additional groups

for each material to be assayed. Keep distilled H2O before the chicks
at all times.

Prepare sufficient basal rachitic ration for the entire feeding period
(60 pounds per 100 birds is ample). Prepare the supplemented rations
at 8-12 day periods. Supplement the basal rachitic ration with corn oil in
a quantity equal to the maximum addition of the oil to be assayed. (This
is the ration to be fed to the negative control group.) Supplement the
basal ration with one or more levels of an assayed cod liver oil, the
level or levels to be so selected that the birds of one group will produce
normal calcification. (These are the rations to be fed to the positive
control group or groups.) Add corn oil to bring the percentage of oil
up to that added to the negative control ration. (These are the rations
to be fed to the other groups.)

On the second day give the groups two 15-minute feedings of their
respective rations. Beginning the third day, feed the rations ad libitum
for 21 days.

Kill the birds, remove the left tibia of each bird, and clear of adhering
tissue. (To facilitate removal of adhering tissue, the bones may be
placed in boiling H:O for not more than two minutes.) If composite
ashing of the tibiae of the groups is to be made, preserve the opposite
tibiae in ethyl alcohol. Number the bones and place in 95% ethyl
alcohol. Crush, wrap in filter paper, and extract the bones for 20 hours
with hot 95% ethyl alcohol, followed by 20 hours with ethyl ether.

(Other solvents may be used for this fat extraction.) Dry in a moisture
oven, and store in a desiccator. Determine the percentage of ash
of the moisture and fat-free bones by igniting individually or by group
composite in a muffle furnace at approximately 850 degrees for one hour.

To obtain vitamin D potency of a product in terms of A. O. A. C.
chick units, use the U. S. Pharmacopoeia “Reference Cod Liver Oil”
in control rations at properly selected levels and compare the resulting
control ash averages with the average produced by the material under
assay. One A. O. A. C. chick unit of vitamin D is equal in biological
activity for the chick to one unit of vitamin D in the U. S. P. Reference
Cod Liver Oil in this method of assay.

A rather complete summary of the results of tests conducted on vitamin D
supplements by the original four-week test, under the percentage guarantee in
force during 1934-1936 is given in the table presented herein. Also included
in the table are results of 19382 and 1988 tests which had been conducted at
such levels that the results could be interpreted on the basis of the 1934
guarantees.

Following the table is an appendix which shows the form in which guarantees
must be expressed for registration and labeling of vitamin supplements in Mary-
land. These forms correspond to those approved by other States carrying on
enforcement work with vitamin D supplements.

Also in the appendix are quoted general recommendations, resolutions, and
definitions of the Association of American Feed Control Officials, Inc., which are
pertinent to the subject of vitamin supplements.

5

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Appendix

1. The following information must be shown on each container of vitamin D
supplements merchandised in the State of Maryland.

(a) Net weight or quantity;
(b) Name of product;

(c) Vitamin D Potency: Guaranteed to contain not less than (?) Associa-
tion of Official Agricultural Chemists’ chick units of vitamin D per
gram:

(d) Name and address of manufacturer or party responsible for sale.

2. The following resolution has been adopted by the Association of American
Feed Control Officials, Inc.

Resolved that any material sold primarily as a vitamin D supplement
for poultry shall be labeled with a minimum guarantee of vitamin D
potency expressed as Association of Official Agricultural Chemists’ chick
units of vitamin D per gram, determined according to the tentative pro-
cedure adopted by the Association of Official Agricultural Chemists, and
assuming that one U. S. P. unit of vitamin D in the U. S. Pharmacopoeia
“Reference Cod Liver Oil” equals one Association of Official Agricultural
Chemists’ chick unit.

3. The following definitions have been adopted as official definitions by the
Association of American Feed Control Officials, Inc.

Cod Liver Oil is the product obtained by extraction of part of the oil
from cod livers.

Sardine Oil or Pilchard Oil is the product obtained by extraction of
part of the oil from the whole Pacific sardine or pilchard or from cannery
refuse of this species of fish.

Salmon Oil is the product obtained by extraction of part of the oil from
the cannery refuse of salmon.

Tuna Oil is the product obtained by extraction of part of the oil from
the cannery refuse of tuna.

Menhaden Oil is the product obtained by extraction of part of the
oil from whole menhaden.

Herring Oil is the product obtained by extraction of part of the oil
from the whole herring or part of the herring.

Salmon Liver Oil is the product obtained by extraction of part of the
oil from salmon livers.

Stearine is the solid material obtained by filtration from an animal
or vegetable oil after chilling or freezing in the process of refining. When
this product is sold as a feed, the term shall be prefixed by the name of
the oil from which it is obtained.

4. The following are tentative definitions of the Association of American Feed
Control Officials, Inc.

Irradiated Yeast is yeast which has been irradiated in order to increase
its anti-rachitic potency and shall contain at least 45 per cent protein
on the moisture free basis. (NOTE—TIf yeast or irradiated yeast consists of
bakers’ type yeast, the term “bakers’ type” shall precede the word yeast;
if it consists of brewers’ type yeast the term “brewers’ type” shall precede
the word yeast.)

11

Cod Liver Oil is the oil obtained from the livers of gadus morrhuae
and/or other species of the family gadidae. It must contain not less than
600 U. S. P. units of vitamin A and not less than 85 Association of Official
Agricultural Chemists’ chick units of vitamin D per gram.

Fortified Cod Liver Oil is cod liver oil mixed with other suitable fish
oils and/or fish oil concentrates so as to contain not less than 400 Associa-
tion of Official Agricultural Chemists’ chick units of vitamin D per gram
and shall be labeled with the minimum guarantee of vitamin A potency
in terms of U. S. P. units of vitamin A per gram.

NOTE—Tentative definitions have received consideration, but are not official. They
are subject to revision before final adoption.