Radial. Phys. Chem. Vol. 42, Nos 46, pp.

605609, 1993
Printed in Great Britain. All rights reserved

0146-5724/93 $6.00 + 0.00

Copyright 1993 Pergamon Press Ltd

RADIATION STERILIZATION OF PHARMACEUTICAL-OPHTHALMIC OINTMENTS

M.L. BOTELHO*, M.C. GODINHO~,M. PARTIDARIO~and M.E. ANDRADE
Physics Department, ICEN, LNETI, E.N. n~10, 2685 Sacavm, Portugal
Laboratrios Pfizer, Apartado 1402, 1012 Lisboa Codex, Portugal

ABSTRACT
The ophthalmic suspension whose active compounds are antibiotic and corticosteroids,
was submitted, in the final package, to radiation in a Cobalt-60 source and their stability
to gamma radiation has been investigated. This procedure alms to substitute the conventional aseptic method of manufacturing and packaging for radiation sterilization. Microbiological studies have been carried out to determinethe minimum absorbed dose achieving
the required Sterility Assurance Level (SAL) of 10_6. The microbiological and product
stability data obtained till the present time, support the use of radiation sterilization.
KEYWORDS
Radiosterilization; pharmaceutical products; stability; microorganism D~jue;SAL.
INTRODUCTION
After several years of being producing ointment and ophthalmic suspension in sterile
conditions by the end of 1991 Pfizer Laboratories S.A. Portugal caine to the decision of
discontinuing the Sterile Area where these ophthalmic products used to be prepared and
filled. Based upon the experience of other laboratories in the same international group,
Pfizer Laboratories decided to sterilize the ointment and the ophthalmic suspension by
gamma radiation.

Although, ophthalmic ointments could be irradiated at 25 kGy, the ophthalmic suspension

have been evidenced physical alterations, namely the colour, through compatibility assays,
performed by Pfizer Laboratories. As it is possible to employ lower doses to sterilize
articles or drugs when the nature of the products is unstable to higher doses, Pfizer
Laboratories and LNETI have iniciated together a study to select an absorbed dose lower
enough to garantee on one hand the stability of the ophthalmic suspension and on the
other one the SAL of 10-6.
MATERIAL
TerraCortrilophthalmic eye/ear suspension (5 ml): oxytetracycline hydrochloride
5 mg/mi; polymyxin B sulphate 10 000 units/mi and hydrocortisone acetate 10 mg/mi;
aluminium tristearate and liquid paraffin vehicle. The product was manufactured under
no sterile conditions, although the GMP rules for pharmaceutical were fullifiled.
605

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M. L. BOTELHO ef a!.

STABILITY STUDIES
Procedures
The present studies include the stability data obtained from samples treated by gamma
radiation with absorbed doses from 0 up to 25 kGy. The irradiation was done in a
Cobalt-60 plant UTR GAMA-Pi with an activity of 7.4 x 10s Bq (July 1992), the dose
uniformity (U) was 1.1. The stability studies were performed by Pfizer Laboratories.

Oxytetracycline Hydrochloride. The oxytetracycline hydrochloride stability was evaluated by using chemical and microbiological methods. The chemical method it was performed by using the caustic degradation method; the oxytetracycline degradation was
measured by spectrophotometry at 353 nm. In the microbiological method the oxytetracycline hydrochloride potency was determined by turbidimetric assay using Kleb3iella
pneiimoniae ATCC 10031 as test organism.
Polymyxin B Sulphate. The polymyxin B sulphate potency was evaluated by the microbiological Cylinder Plate Method using Achromobacter sp. (Pfizer/U.K. strain) as test
organism. The solid medium utilized was Polymyxin Seed Agar.

Hydrocortisone Acetate. The hydrocortisone acetate activity was determined in presence

of tetrazolium blue. The red colour developed was measured by a spectrophotometric
method at 525 nm.
Results
Product colour has changed to dark yellow at 20 and 25 kGy, which leads us to conclude
that doses higher than 20 kGy shall not be carried out.
The results showed in Table 1 present the stability data for the absorbed doses from 0
up to 25 kGy. The active compounds are expressed in a percentage of the label claim.
All the results stayed within the acceptable limits. No degradation products have been
identified.
Table 1

Stability Data at Room Temperature

Absorbed Dose
0 kGy
a.c.

OM

t/m

OCPMHC

106.8 102.8 96.8 103.2

101.3 100.3 97.0 104.2

12

100.4 100.5 98.2 100.5

a.c

active compound; t/m

10 kGy
OMOCPM
93.8 96.2

HC

99.4 101.3

101.6 98.2 94.1 111.3

97.8 99.2 100.7

96.0

time/month; OM

OC

Oxytetracycline Chemical test; PM

HC

Hydrocortisone acetate chemical test.

15 kGy

not determined due to colour change.

OM

OC

PM

20 kGy
HC

96.6 102.8 105.9 100.7

99.0 96.4

94.8 108.0

104.6 99.7

94.6 96.0

OMOCPMHC
97.0 91.4 96.9 101.3

Oxytetracycline Microbiological test;

Polymyxin B sulphate Microbiological test;

25 kGy
OMOCPMHC
96.0 96.0 95.2 97.3

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Meeling on Radiation Processing

607

MICROBIOLOGICAL STUDIES
Procedures
To validate the lower doses efficacy, it was necessary to quantify the bioburden and
determine the radiation resistance of the natural contaminants of the product prior to
sterilization. As the frequency of natural contaminants was too low, the minimum absorbed dose (Dmin) was estimated based on B. pumilus Dvalue as it represents the highest
Dvaiue Covering approximately 99.8% of radioresistance frequency of microbial population
(AAMI, 1984). The influence of ophthalmic suspension on the B. pumilu3 Dvaiue was
studied. The microbiological studies were done by LNETI except when stated otherwise.

Bioburden (N0). Bioburden was determined, following the USP Federal Register Sterility
test for TerramycinCortril ophtalmic suspension (USP XXII, 1990), in three different
batchs, prior to sterilization. A sample of 100 ophthalmic suspension tubes was taken at
random, from each batch, by using an aleatory listing generated by an automatic statistic
program. The total of each tube (5 ml) was splitted in two equal parts and suspended
respectively in 900 ml 100 ml of USP Fluid Thioglycollate and USP Soybean Casein
Digest medium. The incubation period was extended up to 14 days.
This method was validated using spores of Bacillus sp. to determine the efficacy and
reproducibility of the bioburden recovery method. The tubes collected were distributed
randomly to both laboratories, Pfizer Laboratories and LNETI, with the purpose of performing a control for false positives and introducing these tests on a routine basis in the
company, to calculate the maximum microbial contamination in order to meet a Sterility
Assurance Level of 10-6 with the minimum absorbed dose determined during this validation.
The natural contaminants were identified by using the API system for bacteria whereas
the moulds were identified to genus level by using the identification keys provided by
Onions et al. (1981), Barnett (1969) and Ellis (1971).

Dvaiue. Dvaiue determination was based on quantal estimates of survivor in fraction positive
range (Pflug et al., 1983). Five to ten replica of pre-sterilized ophtalmic suspension tubes

were contaminated with a known number of natural contaminants (10~to 10~c.f.u.) and
afterwards irradiated to sub-sterilized doses. Irradiation was done at a define place in
gamma plant UTR GAMAPi. Dose Rate =2.6 kGy.h (August 1991) determined by
Fricke dosimeter and measured by ionizing chamber. U=1.017.
Influence of the Ophthalmic Suspension on B. Pumilus P2601 Dvsiue.
A batch of test
pieces of B. purnilus P2601, approx. 108 c.f.u., was prepared by using the procedure
developed by E. Christensen (Christensen et aL, 1964), to study the influence of the ophthalmic suspension on the Dvaiue of this standard strain. Two inactivation curves were
built: one corresponding to the test pieces irradiated with the doses from 5 to 20 kGy
plated on Tryptone Glucose Yeast Agar and another one obtained from the test pieces
introduced whithin the product homogeneized and irradiated with 5 to 16 kGy plated in
the same medium. Irradiation was done in the local above described.
Results
The frequency of contaminated
items Most
(N0) shown
in Tablecontaminants
II allows to estimate
mean
2 (Sokal, 1981).
of the natural
showed a agrowth
incidence of 1.67 x10

RPC 42-4/6-D

M. L.

608

BOTELHO et a!.

after 7 days. The microorganism test (spores of Bacillus sp.) in the same conditions was
observed at 24-48h. All the microorganisms showed Dyajues lower than B. pumilus E601
Dvalue as it could be seen in Table 3.
Table 2. Frequencies of Contaminated Items
Item
Batch

no contaminated

contaminated

TOTAL

1
2

96
100

100
100

99

100

TOTAL

295

300

Table 3. Natural Contaminants Microorganisms Isolated and Dvatues

Microorganisms
Batch

Genus

Staphylococcus sp.

0.22 kGy

Flavobacterium sp.

0.19 kGy
2.26 kGy
2.09 kGy

Stachybotrys sp.
Cladosporium sp.

Dyajue

Cladosporium sp.

1.43 kGy

As presented in Fig. 1 the slope of both inactivation curves showed no significant differences which suggest that the ophthalmic suspension TerraCortril did not affect the
resistance (Dvaiue) of the B. pumilus E601 to radiation.

Inactivation Curve

10

B.pumilizs E601 vs Dose

0.0

5.0

10.0

15.0

20.0

25.0 (kGy)

Absorbed Dose
oDvotue 3.3 kGy
oDvolue 3.2 kGy

Fig. 1. Inactivation curves of B. pumilus

8th International Meeting on Radiation Processing

609

CONCLUSIONS
Although a slight darkening having been observed the produt was stable after being
exposed to 10 and 15 kGy and after storage for 12 months at room temperature.
The product bioburden prior to sterilization was of a low degree demonstrated by the
mean incidence as well as the characteristics of microbial growth. Natural contaminants
Dvaiue showed to be lower than B. pumilus E601 ~
in the circumstances studied. No
significant influence of Terra-Cortril ophthalmic suspension in the resistance of B. pumilus
E601 was found. Due to the low frequency of natural contaminants a sterilization dose
(Dmin) of 13 kGy was determined based on the Dyajue spores of B. pumilus E601, the mean
incidence of contaminated items and the SAL of 10_6 (Botelho ci. al., 1988).
Nevertheless, to maintain this sterilization dose microbiological control should be performed, in all batchs prior to sterilization, to confirm there are no changes of the mean
incidence of N0.

ACKNOWLEDGMENTS
The authors thank Helena Marcos and Pedro Pereira for their assistance in experimental
work and to Luisa Oliveira for typing the text.

REFERENCES
AAMI (1984). Process Control Guidelines for Gamma Radiation Sterilization of Medical
Devices. AAMI RS-3/84, USA.
Barnett, H.L. (1969). Imperfect Fungi. Burgess Publishing Company, Minneapolis.
Boteiho, M.L., et al. (1988). Searching for a Strategy to Gamma Sterilize Portuguese
Cork Stoppers Preliminary Studies on Bioburden, Radioresistance and Sterility As

surance Level. Rad. Phys. Chem., ~1, 775781.

Christensen, E.A. ci al. (1964). Irradiation of Dried Bacteria and Bacterial Spores by
Means of Ionizing Radiation. Ada Path. Microb. Scand., fi0253264.
Ellis, M.B. (1971). Dematiaceous Hyphomycetes. Institute Kew, Surrey, England
Onions, A.H.S., et al. (1981). Smiths Introduction to Industrial Mycology. Edward
Arnold, London.

Pharmacopeia of the United States of America. USP XXII, The United States Pharmacopeiai Convention, Inc. USA.
Pfiug, I.J. ei al. (1983). Principles of Thermal Destruction of Microorganisms. In: Desinfection, Sterilization and Preservation (S.B. Block, ed.), pp.795803. Lea & Febiger,
Philadelphia.
Soka.l, R.R. and F.J. Rohlf (1981). Introduction to Probability Distributions: Binomial
and Poisson. In: Biometrp (R.R. Sokal and F.J. Rohif), pp.7082. 2~Ed., W.H.
Freeman and Company, New York.