Packaging and storage—
Preserve in the original package under the conditions recommended on the label, and protect it from light, toxic substances, excessive heat, and moisture. The packaging and container material shall be such that it does not adversely affect the performance of the article used as directed in the labeling.
Expiration date—
The expiration date is determined on the basis of stability studies and is not less than 18 months from the date of manufacture, the date of manufacture being the date on which the first determination of the total viable spore count was made.
Labeling—
Label it to state that it is a Biological Indicator for Ethylene Oxide Sterilization, Paper Carrier; to indicate its D value, the method used to determine such D value, i.e., by spore count or fraction negative procedure after graded exposures to the sterilization conditions; the survival time and kill time under specified sterilization conditions stated on the label; its particular total viable spore count, with a statement that such count has been determined after preliminary heat treatment; and its recommended storage conditions. State in the labeling the size of the paper carrier, the strain and ATCC number from which the spores were derived, and instructions for spore recovery and for safe disposal of the indicator. Indicate in the labeling that the stated D value is reproducible only under the exact conditions under which it was determined, that the user would not necessarily obtain the same result, and that the user should determine the suitability of the biological indicator for the particular use.
Identification—
The biological indicator organism complies substantially with the morphological, cultural, and biochemical characteristics of the strain of
Bacillus subtilis, ATCC No. 9372, designated subspecies
niger: under microscopic examination it consists of Gram-positive rods of width 0.7 to 0.8 µm, and length 2 to 3 µm; the endospores are oval and central and the cells are not swollen; when incubated aerobically in appropriate media at 30
to 35
, growth occurs within 24 hours, and similar inoculated media incubated concomitantly at 55
to 60
show no evidence of growth in the same period; agar colonies have a dull appearance and may be cream or brown-colored; when incubated in nutrient broth it develops a pellicle, and shows little or no turbidity; when examined under conventional biochemical tests for microbial characterization, it develops a black pigment with tyrosine, it liquefies gelatin, utilizes citrate but not propionate or hippurate, reduces nitrate, and hydrolyzes both starch and glucose with no gas production; it shows a positive catalase reaction and gives a positive result with the Voges-Proskauer test.
Resistance performance tests—
D value—
Proceed as directed for the relevant procedure for
D value under
Biological Indicators—
Resistance Performance Tests 
55
. The requirements of the test are met if the determined
D value is within 20% of the labeled
D value for the selected sterilizing temperature and if the confidence limits of the estimate are within 10% of the determined
D value.
Survival time and kill time—
Proceed as directed for
Survival Time and Kill Time in the section
Ethylene Oxide Sterilization,
Paper Carrier, under
Biological Indicators—
Resistance Performance Tests 55. The requirements of the test are met if all of the specimens subjected to the ethylene oxide sterilization conditions for the survival time show evidence of growth, while none of the specimens subjected to the ethylene oxide sterilization conditions for the kill time shows evidence of growth. If for either the survival time test or the kill time test, not more than 1 specimen out of both groups fails the survival requirement or the kill requirement (whichever is applicable), continue the corresponding test with 4 additional groups, each consisting of 20 specimens, according to the procedure described. If all of the additional specimens subjected to ethylene oxide sterilization meet either the survival requirement for the survival time test or the kill requirement for the kill time test, whichever is applicable, the requirements of the test are met.
Total viable spore count—
Follow the procedure for
Total Viable Spore Count in the section
Ethylene Oxide Sterilization,
Paper Carrier, under
Biological Indicators—
Resistance Performance Tests 55. The requirements of the test are met if the log average number of viable spores per carrier is not less than 0.3 log of the labeled spore count per carrier and does not exceed the log labeled spore count per carrier by 0.48.
Purity—
Presence of contamination by other microorganisms—
By examination of the spores on a suitable plate culture medium, there is no evidence of contamination with other microorganisms.
Disposal—
Prior to destruction or discard, sterilize it by steam at 121
for not less than 30 minutes, or by not less than an equivalent method recommended by the manufacturer. This includes a strip used in test procedures for strips themselves.
2), and