Q4B Evaluation and Recommendation of 

Pharmacopoeial Texts for Use in the ICH Regions 

Annex 8: Sterility Test General Chapter 

This draft guidance, when finalized, will represent the Food and Drug Administration's (FDA's) 

current thinking on this topic. It does not create or confer any rights for or on any person and 

does not operate to bind FDA or the public. You can use an alternative approach if the approach 

satisfies the requirements of the applicable statutes and regulations. If you want to discuss an 

alternative approach, contact the FDA staff responsible for implementing this guidance. If you 

cannot identify the appropriate FDA staff, call the appropriate number listed on the title page of 

this guidance. 

For questions regarding this draft document contact (CDER) Robert King 301-796-1242, 

or (CBER) Christopher Joneckis 301-827-0373. 
 

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3 INTERNATIONAL CONFERENCE ON HARMONISATION OF TECHNICAL 

4 REQUIREMENTS FOR REGISTRATION OF PHARMACEUTICALS FOR HUMAN USE 

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8 DRAFT CONSENUS GUIDELINE
 

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14 EVALUATION AND RECOMMENDATION OF 

PHARMACOPOEIAL TEXTS FOR USE IN THE ICH REGIONS 

16 ON 

17 STERILITY TEST GENERAL CHAPTER 

18 

19 Q4B ANNEX 8 

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22 

23 

24 Current Step 2 Version 1 

dated 13 November 2008 

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At Step 2 of the ICH Process, a consensus draft text or guideline, agreed by the appropriate ICH 

36 Expert Working Group, is transmitted by the ICH Steering Committee to the regulatory 

37 authorities of the three ICH regions (the European Union, Japan and the USA) for internal and 

38 external consultation, according to national or regional procedures. 
 

 

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50 Q4B Annex 8 

51 Document History 

52 

53 Current Step 2 version 

54 

Code History Date 

Q4B Annex 8 Approval by the Steering Committee under Step 2 and release 

for public consultation. 

13 November 

2008 

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59 EVALUATION AND RECOMMENDATION OF 

60 PHARMACOPOEIAL TEXTS FOR USE IN THE ICH REGIONS 

61 ON 

62 STERILITY TEST GENERAL CHAPTER 

63 

64 Q4B Annex 8 

65 

66 Draft ICH Consensus Guideline 

67 Released for Consultation on 13 November 2008, at Step 2 of the ICH Process 

68 

69 

70 TABLE OF CONTENTS 

71 

72 

73 1. INTRODUCTION......................................................................................................1 

74 

75 2. Q4B OUTCOME........................................................................................................1 

76 

77 2.1 Analytical Procedures................................................................................................1 

78 

79 2.2 Acceptance Criteria....................................................................................................1 

80 

81 3. TIMING OF ANNEX IMPLEMENTATION...........................................................1 

82 

83 4. CONSIDERATIONS FOR IMPLEMENTATION.................................................1 

84 

85 4.1 General Consideration................................................................................1 

86 

87 4.2 FDA Consideration....................................................................................2 

88 

89 4.3 EU Consideration......................................................................................2 

90 

91 4.4 MHLW Consideration.................................................................................2 

92 

93 5. REFERENCES USED FOR THE Q4B EVALUATION.......................................2 

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97 EVALUATION AND RECOMMENDATION OF 

98 PHARMACOPOEIAL TEXTS FOR USE IN THE ICH REGIONS 

99 ON 

100 STERILITY TEST GENERAL CHAPTER

101 

102 Q4B Annex 8 103

104 

105 1. INTRODUCTION 106 

107 This annex is the result of the Q4B process for the Sterility Test General Chapter. 

108 

109 The proposed texts were submitted by the Pharmacopoeial Discussion Group (PDG). 

110 

111 2. Q4B OUTCOME 

112 

113 2.1 Analytical Procedures 114 

115 The ICH Steering Committee, based on the evaluation by the Q4B Expert Working 

116 Group (EWG), recommends that the official pharmacopoeial texts, Ph.Eur. 2.6.1. 

117 Sterility, JP 4.06 Sterility Test, and USP <71> Sterility Tests, can be used as 

118 interchangeable in the ICH regions subject to the conditions detailed below. Testing 

119 conditions for medical devices, such as sutures, are outside the scope of the ICH 

120 recommendation. 

121 

122 2.1.1 Local texts identified by the black diamond symbol are not considered 

123 interchangeable in all regions. 

124 

125 2.1.2 Diluting and rinsing fluids should not have antibacterial or antifungal 

126 properties if they are to be considered suitable for dissolving, diluting, or 

127 rinsing an article under test for sterility. 

128 

129 2.2 Acceptance Criteria 130 

131 The acceptance criteria are harmonized between the three pharmacopoeias. 

132 

133 3. TIMING OF ANNEX IMPLEMENTATION 134 

135 When this annex is implemented (incorporated into the regulatory process at ICH Step 5) in a 

136 region, it can be used in that region. Timing might differ for each region. 

137 

138 4. CONSIDERATIONS FOR IMPLEMENTATION 139 

140 4.1 General Consideration 141 

142 When sponsors or manufacturers change their existing methods to the implemented 

143 Q4B-evaluated pharmacopoeial texts that are referenced in Section 2.1 of this annex, 

144 any change notification, variation, and/or prior approval procedures should be handled 

145 in accordance with established regional regulatory mechanisms pertaining to 

146 compendial changes. 147 148 

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149 Sterility Test General Chapter 150 

151 

152 4.2 FDA Consideration 

153 

154 Based on the recommendation above, and with reference to the conditions set forth in 

155 this annex, the pharmacopoeial texts referenced in Section 2.1 of this annex can be 

156 considered interchangeable. However, FDA might request that a company 

157 demonstrate that the chosen method is acceptable and suitable for a specific material 

158 or product, irrespective of the origin of the method. 

159 

160 4.3 EU Consideration 161 

162 For the European Union, the monographs of the Ph. Eur. have mandatory 

163 applicability. Regulatory authorities can accept the reference in a marketing 

164 authorisation application, renewal or variation application citing the use of the 

165 corresponding text from another pharmacopoeia as referenced in Section 2.1, in 

166 accordance with the conditions set out in this annex, as fulfilling the requirements for 

167 compliance with the Ph. Eur. Chapter, Sterility: 2.6.1., on the basis of the declaration 

168 of interchangeability made above. 169 

170 4.4 MHLW Consideration 171 

172 The pharmacopoeial texts referenced in Section 2.1 of this annex can be used as 

173 interchangeable in accordance with the conditions set out in this annex. Details of 

174 implementation requirements will be provided in the notification by MHLW when this 

175 annex is implemented. 

176 

177 

178 

179 5. REFERENCES USED FOR THE Q4B EVALUATION 180 

181 5.1 The PDG Stage 5B sign-off document: Japanese Pharmacopoeial Forum, Volume 

182 16, number 4 (December 2007). 183 

184 5.2 The pharmacopoeial references for Sterility Test for this annex are: 

185 

186 5.2.1 European Pharmacopoeia (Ph. Eur.): 

187 Supplement 6.3 (official in January 2009), Sterility (reference 01/2009:20601). 

188 

189 5.2.2 Japanese Pharmacopoeia (JP): 

190 The 4.06 Sterility Test will be made official via Ministerial Notification (March 

191 2009). The draft English version of the JP text is appended. 

192 

193 5.2.3 United States Pharmacopeia (USP): 

194 <71> Sterility Tests as presented in Pharmacopeial Forum Volume 34(6), 

195 Interim Revision Announcement No. 6, official December 1, 2008.
 

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197 English text as provided to the Q4B EWG 

198 This text below has been provided by the Ministry of Health, Labour and Welfare (MHLW) and 

199 represents an English translation of a Ministerial Notification to be published in March 2009. 

200 

201 This test is harmonized with the European Pharmacopoeia and the U. S. Pharmacopeia. 

202 

203 The test is applied to substances, preparations or articles which, according to the 

204 Pharmacopoeia, are required to be sterile. However, a satisfactory result only indicates that no 

205 contaminating micro-organism has been found in the sample examined in the conditions of the 

206 test. 

207 1. Precautions against microbial contamination 

208 The test for sterility is carried out under aseptic conditions. In order to achieve such conditions, 

209 the test environment has to be adapted to the way in which the sterility test is performed. The 

210 precautions taken to avoid contamination are such that they do not affect any micro-organisms 

211 which are to be revealed in the test. The working conditions in which the tests are performed are 

212 monitored regularly by appropriate sampling of the working area and by carrying out appropriate 

213 controls. 

214 2. Culture media and incubation temperatures 

215 2.1. Introduction 

216 Media for the test may be prepared as described below, or equivalent commercial media may 

217 be used provided that they comply with the growth promotion test. 

218 The following culture media have been found to be suitable for the test for sterility. Fluid 

219 thioglycollate medium is primarily intended for the culture of anaerobic bacteria; however, it will 

220 also detect aerobic bacteria. Soya-bean casein digest medium is suitable for the culture of both 

221 fungi and aerobic bacteria. 

222 

223 2.2. Fluid thioglycollate medium 

224 Fluid thioglycollate medium 

225 L-Cystine 0.5 g 

226 Agar 0.75 g 

227 Sodium chloride 2.5 g 

228 Glucose monohydrate/anhydrous 5.5 / 5.0 g 

229 Yeast extract (water-soluble) 5.0 g 

230 Pancreatic digest of casein 15.0 g 

231 Sodium thioglycollate or 0.5 g 

232 Thioglycollic acid 0.3 mL 

233 Resazurin sodium solution (1 in 1000), freshly prepared 1.0 mL 

234 Water 1 000 mL 

235 (pH after sterilization 7.1 ± 0.2) 

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236 Mix the L-cystine, agar, sodium chloride, glucose, water-soluble yeast extract and pancreatic 

237 digest of casein with water, and heat until solution is effected. Dissolve the sodium thioglycollate 

238 or thioglycollic acid in the solution and, if necessary, add sodium hydroxide TS so that, after 

239 sterilization, the solution will have a pH of 7.1 ± 0.2. If filtration is necessary, heat the solution 

240 again without boiling and filter while hot through moistened filter paper. Add the resazurin 

241 sodium solution (1 in 1000), mix and place the medium in suitable vessels which provide a ratio 

242 of surface to depth of medium such that not more than the upper half of the medium has 

243 undergone a colour change indicative of oxygen uptake at the end of the incubation period. 

244 Sterilize using a validated process. If the medium is stored, store at a temperature between 2 °C 

245 and 25 °C in a sterile, airtight container. If more than the upper one-third of the medium has 

246 acquired a pink colour, the medium may be restored once by heating the containers in a water247 bath or in free-flowing steam until the pink colour disappears and cooling quickly, taking care to 

248 prevent the introduction of non-sterile air into the container. Do not use the medium for a longer 

249 storage period than has been validated. 

250 Fluid thioglycollate medium is to be incubated at 30-35 °C. 

251 For products containing a mercurial preservative that cannot be tested by the membrane252 filtration method, fluid thioglycollate medium incubated at 20-25 °C may be used instead of soya253 bean casein digest medium provided that it has been validated as described in growth promotion 

254 test. 

255 

256 Where prescribed or justified and authorized, the following alternative thioglycollate medium 

257 might be used. Prepare a mixture having the same composition as that of the fluid thioglycollate 

258 medium, but omitting the agar and the resazurin sodium solution (1 in 1000), sterilize as directed 

259 above. The pH after sterilization is 7.1 ± 0.2. Heat in a water bath prior to use and incubate at 30­

260 35 °C under anaerobic conditions. 

261 

262 2.3. Soya-bean casein digest medium 

263 Soya-bean casein digest medium 

264 Pancreatic digest of casein 17.0 g 

265 Papaic digest of soya-bean meal 3.0 g 

266 Sodium chloride 5.0 g 

267 Dipotassium hydrogen phosphate 2.5 g 

268 Glucose monohydrate/anhydrous 2.5 / 2.3 g 

269 Water 1 000 mL 

270 (pH after sterilization 7.3 ± 0.2) 

271 Dissolve the solids in water, warming slightly to effect solution. Cool the solution to room 

272 temperature. Add sodium hydroxide TS, if necessary, so that after sterilization the solution will 

273 have a pH of 7.3 ± 0.2. Filter, if necessary, to clarify, distribute into suitable vessels and sterilize 

274 using a validated process. Store at a temperature between 2 °C and 25 °C in a sterile well-closed 

275 container, unless it is intended for immediate use. Do not use the medium for a longer storage 

276 period than has been validated. 

277 Soya-bean casein digest medium is to be incubated at 20-25 °C. 

278 3. Suitability of the culture medium 

279 The media used comply with the following tests, carried out before or in parallel with the test 

280 on the product to be examined. 

281 Sterility 

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282 Incubate portions of the media for 14 days. No growth of micro-organisms occurs. 

283 Growth promotion test of aerobes, anaerobes and fungi 

284 Test each batch of ready-prepared medium and each batch of medium prepared either from 

285 dehydrated medium or from ingredients. Suitable strains of micro-organisms are indicated in 

286 Table 4.06 -1. 

287 Inoculate portions of fluid thioglycollate medium with a small number (not more than 100 

288 CFU) of the following micro-organisms, using a separate portion of medium for each of the 

289 following species of micro-organism: Clostridium sporogenes, Pseudomonas aeruginosa, 

290 Staphylococcus aureus. 

291 Inoculate portions of soya-bean casein digest medium with a small number (not more than 

292 100 CFU) of the following micro-organisms, using a separate portion of medium for each of the 

293 following species of micro-organism: Aspergillus niger, Bacillus subtilis, Candida albicans. 

294 Incubate for not more than 3 days in the case of bacteria and not more than 5 days in the case 

295 of fungi. 

296 Seed lot culture maintenance techniques (seed-lot systems) are used so that the viable micro297 organisms used for inoculation are not more than five passages removed from the original master 

298 seed-lot. 

299 The media are suitable if a clearly visible growth of the micro-organisms occurs 

300 Table 4.06 -1 — Strains of the test micro-organisms suitable for use in the 

301 Growth Promotion Test and the Method suitability Test 

302 Aerobic bacteria 

303 Staphylococcus aureus 

304 NCIMB 9518 

305 Bacillus subtilis 

306 Pseudomonas aeruginosa 

307 Anaerobic bacterium 

308 Clostridium sporogenes 

309 ATCC 11437 

310 Fungi 

311 Candida albicans 

312 Aspergillus niger 

313 4. Method suitability test 

ATCC 6538, NBRC 13276, CIP 4.83, NCTC 10788, 

ATCC 6633, NBRC 3134, CIP 52.62, NCIMB 8054 

ATCC 9027, NBRC 13275, NCIMB 8626, CIP 82.118 

ATCC 19404, NBRC 14293, CIP 79.3, NCTC 532 or 

ATCC 10231, NBRC 1594, IP 48.72, NCPF 3179 

ATCC 16404, NBRC 9455, IP 1431.83, IMI 149007 

314 Carry out a test as described below under Test for sterility of the product to be examined using 

315 exactly the same methods except for the following modifications. 

316 Membrane filtration 

317 After transferring the content of the container or containers to be tested to the membrane add 

318 an inoculum of a small number of viable micro-organisms (not more than 100 CFU) to the final 

319 portion of sterile diluent used to rinse the filter. 

320 Direct inoculation 

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321 After transferring the contents of the container or containers to be tested to the culture medium 

322 add an inoculum of a small number of viable micro-organisms (not more than 100 CFU) to the 

323 medium. 

324 In both cases use the same micro-organisms as those described above under Growth promotion 

325 test of aerobes, anaerobes and fungi. Perform a growth promotion test as a positive control. 

326 Incubate all the containers containing medium for not more than 5 days. 

327 If clearly visible growth of micro-organisms is obtained after the incubation, visually 

328 comparable to that in the control vessel without product, either the product possesses no 

329 antimicrobial activity under the conditions of the test or such activity has been satisfactorily
 

330 eliminated. The test for sterility may then be carried out without further modification. 

331 If clearly visible growth is not obtained in the presence of the product to be tested, visually
 

332 comparable to that in the control vessels without product, the product possesses antimicrobial 

333 activity that has not been satisfactorily eliminated under the conditions of the test. Modify the 

334 conditions in order to eliminate the antimicrobial activity and repeat the method suitability test. 

335 This method suitability is performed: 

336 a) when the test for sterility has to be carried out on a new product; 

337 b) whenever there is a change in the experimental conditions of the test. 

338 The method suitability may be performed simultaneously with the Test for sterility of the 

339 product to be examined. 

340 5. Test for sterility of the product to be examined 

341 5.1. Introduction 

342 The test may be carried out using the technique of membrane filtration or by direct inoculation 

343 of the culture media with the product to be examined. Appropriate negative controls are included. 

344 The technique of membrane filtration is used whenever the nature of the product permits, that is, 

345 for filterable aqueous preparations, for alcoholic or oily preparations and for preparations 

346 miscible with or soluble in aqueous or oily solvents provided these solvents do not have an 

347 antimicrobial effect in the conditions of the test. 

348 5. 2. Membrane filtration 

349 Use membrane filters having a nominal pore size not greater than 0.45 μm whose effectiveness 

350 to retain micro-organisms has been established. Cellulose nitrate filters, for example, are used for 

351 aqueous, oily and weakly alcoholic solutions and cellulose acetate filters, for example, for 

352 strongly alcoholic solutions. Specially adapted filters may be needed for certain products, e.g. for 

353 antibiotics. 

354 The technique described below assumes that membranes about 50 mm in diameter will be 

355 used. If filters of a different diameter are used the volumes of the dilutions and the washings 

356 should be adjusted accordingly. The filtration apparatus and membrane are sterilized by 

357 appropriate means. The apparatus is designed so that the solution to be examined can be 

358 introduced and filtered under aseptic conditions; it permits the aseptic removal of the membrane 

359 for transfer to the medium or it is suitable for carrying out the incubation after adding the medium 

360 to the apparatus itself. 

361 Aqueous solutions 

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362 If appropriate, transfer a small quantity of a suitable, sterile diluent such as a 1 g / L neutral 

363 solution of meat or casein peptone pH 7.1 ± 0.2 onto the membrane in the apparatus and filter. 

364 The diluent may contain suitable neutralizing substances and/or appropriate inactivating 

365 substances for example in the case of antibiotics. 

366 Transfer the contents of the container or containers to be tested to the membrane or 

367 membranes, if necessary after diluting to the volume used in the method suitability test with the 

368 chosen sterile diluent but in any case using not less than the quantities of the product to be 

369 examined prescribed in Table 4.06-2. Filter immediately. If the product has antimicrobial 

370 properties, wash the membrane not less than three times by filtering through it each time the 

371 volume of the chosen sterile diluent used in the method suitability test. Do not exceed a washing 

372 cycle of 5 times 100 mL per filter, even if during method suitability it has been demonstrated that 

373 such a cycle does not fully eliminate the antimicrobial activity. Transfer the whole membrane to 

374 the culture medium or cut it aseptically into two equal parts and transfer one half to each of two 

375 suitable media. Use the same volume of each medium as in the method suitability test. 

376 Alternatively, transfer the medium onto the membrane in the apparatus. Incubate the media for 

377 not less than 14 days. 

378 Table 4.06-2 — Minimum quantity to be used for each medium 

Quantity per container 

Minimum quantity to be used for each 

medium unless otherwise justified and 

authorised 

Liquids 

– less than 1 mL: The whole contents of each container 

– 1-40 mL: Half the contents of each container but not 

less than 1 mL 

– greater than 40 mL and not greater than 100 20 mL 

mL 

– greater than 100 mL : 10 per cent of the contents of the container 

but not less than 20 mL 

Antibiotic liquids 1 mL 

Insoluble preparations, creams and ointments to 

be suspended or emulsified 

Use the contents of each container to provide 

not less than 200 mg 

Solids 

– less than 50 mg The whole contents of each container 

– 50 mg or more but less than 300 mg Half the contents of each container but not 

less than 50 mg 

– 300 mg – 5 g 150 mg 

– greater than 5 g 500 mg 

379 Soluble solids 

380 Use for each medium not less than the quantity prescribed in Table 4.06-2 of the product 

381 dissolved in a suitable solvent such as the solvent provided with the preparation, water for 

382 injection, saline or a 1 g / L neutral solution of meat or casein peptone and proceed with the test 

383 as described above for aqueous solutions using a membrane appropriate to the chosen solvent. 

384 Oils and oily solutions 

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385 Use for each medium not less than the quantity of the product prescribed in Table 4.06-2. Oils 

386 and oily solutions of sufficiently low viscosity may be filtered without dilution through a dry 

387 membrane. Viscous oils may be diluted as necessary with a suitable sterile diluent such as 

388 isopropyl myristate shown not to have antimicrobial activity in the conditions of the test. Allow 

389 the oil to penetrate the membrane by its own weight then filter, applying the pressure or suction 

390 gradually. Wash the membrane at least three times by filtering through it each time about 100 mL 

391 of a suitable sterile solution such as 1 g / L neutral meat or casein peptone containing a suitable 

392 emulsifying agent at a concentration shown to be appropriate in the method suitability of the test, 

393 for example polysorbate 80 at a concentration of 10 g / L. Transfer the membrane or membranes 

394 to the culture medium or media or vice versa as described above for aqueous solutions, and 

395 incubate at the same temperatures and for the same times. 

396 Ointments and creams 

397 Use for each medium not less than the quantities of the product prescribed in Table 4.06-2. 

398 Ointments in a fatty base and emulsions of the water-in-oil type may be diluted to 1 per cent in 

399 isopropyl myristate as described above, by heating, if necessary, to not more than 40 °C. In 

400 exceptional cases it may be necessary to heat to not more than 44 °C. Filter as rapidly as possible 

401 and proceed as described above for oils and oily solutions. 

402 5.3. Direct inoculation of the culture medium 

403 Transfer the quantity of the preparation to be examined prescribed in Table 4.06-2 directly into 

404 the culture medium so that the volume of the product is not more than 10 per cent of the volume 

405 of the medium, unless otherwise prescribed. 

406 If the product to be examined has antimicrobial activity, carry out the test after neutralising this 

407 with a suitable neutralising substance or by dilution in a sufficient quantity of culture medium. 

408 When it is necessary to use a large volume of the product it may be preferable to use a 

409 concentrated culture medium prepared in such a way that it takes account of the subsequent 

410 dilution. Where appropriate the concentrated medium may be added directly to the product in its 

411 container. 

412 Oily liquids 

413 Use media to which have been added a suitable emulsifying agent at a concentration shown to 

414 be appropriate in the method suitability of the test, for example polysorbate 80 at a concentration 

415 of 10 g / L. 

416 Ointments and creams 

417 Prepare by diluting to about 1 in 10 by emulsifying with the chosen emulsifying agent in a 

418 suitable sterile diluent such as a 1 g / L neutral solution of meat or casein peptone. Transfer the 

419 diluted product to a medium not containing an emulsifying agent. 

420 Incubate the inoculated media for not less than 14 days. Observe the cultures several times 

421 during the incubation period. Shake cultures containing oily products gently each day. However 

422 when fluid thioglycollate medium is used for the detection of anaerobic micro-organisms keep 

423 shaking or mixing to a minimum in order to maintain anaerobic conditions. 

424 6. Observation and interpretation of results 

425 At intervals during the incubation period and at its conclusion, examine the media for 

426 macroscopic evidence of microbial growth. If the material being tested renders the medium turbid 

427 so that the presence or absence of microbial growth cannot be readily determined by visual 

428 examination, 14 days after the beginning of incubation transfer portions (each not less than 1 mL) 

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429 of the medium to fresh vessels of the same medium and then incubate the original and transfer 

430 vessels for not less than 4 days. 

431 If no evidence of microbial growth is found, the product to be examined complies with the test 

432 for sterility. If evidence of microbial growth is found the product to be examined does not comply
 

433 with the test for sterility, unless it can be clearly demonstrated that the test was invalid for causes 

434 unrelated to the product to be examined. 

435 The test may be considered invalid only if one or more of the following conditions are fulfilled: 

436 a) the data of the microbiological monitoring of the sterility testing facility show a fault; 

437 b) a review of the testing procedure used during the test in question reveals a fault; 

438 c) microbial growth is found in the negative controls; 

439 d) after determination of the identity of the micro-organisms isolated from the test, the growth 

440 of this species or these species may be ascribed unequivocally to faults with respect to the 

441 material and/or the technique used in conducting the sterility test procedure. 

442 If the test is declared to be invalid it is repeated with the same number of units as in the 

443 original test. 

444 If no evidence of microbial growth is found in the repeat test the product examined complies 

445 with the test for sterility. If microbial growth is found in the repeat test the product examined 

446 does not comply with the test for sterility. 

447 7. Application of the test to parenteral preparations, ophthalmic and other non-injectable 

448 preparations required to comply with the test for sterility 

449 When using the technique of membrane filtration, use, whenever possible, the whole contents 

450 of the container, but not less than the quantities indicated in Table 4.06-2, diluting where 

451 necessary to about 100 mL with a suitable sterile solution, such as 1 g / L neutral meat or casein 

452 peptone. 

453 When using the technique of direct inoculation of media, use the quantities shown in 

454 Table 4.06-2, unless otherwise justified and authorised. The tests for bacterial and fungal sterility 

455 are carried out on the same sample of the product to be examined. When the volume or the 

456 quantity in a single container is insufficient to carry out the tests, the contents of two or more 

457 containers are used to inoculate the different media. 

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458 8. Minimum number of items to be tested 

459 The minimum number of items to be tested in relation to the size of the batch is given in Table 

460 4.06-3. 

461 Table 4.06-3. Minimum number of items to be tested 

Number of items in the batch* 

Minimum number of items to be tested for each 

medium, unless otherwise justified and 

authorised** 

Parenteral preparations 

– Not more than 100 containers 

– More than 100 but not more than 500 

containers 

– More than 500 containers 

10 per cent or 4 containers whichever is the 

greater 

10 containers 

2 per cent or 20 containers (10 containers for 

large-volume parenterals) whichever is the less 

Ophthalmic and other non-injectable 

preparations 

– Not more than 200 containers 5 per cent or 2 containers whichever is the greater 

– More than 200 containers 10 containers 

– If the product is presented in the form of singledose containers, apply the scheme shown above 

for preparations for parenteral use 

Bulk solid products 

– Up to 4 containers 

– More than 4 containers but not more than 

50 containers 

– More than 50 containers 

Each container 

20 per cent or 4 containers whichever is the 

greater 

2 per cent or 10 containers whichever is the 

greater 

462 * If the batch size is not known, use the maximum number of items prescribed 

463 **If the contents of one container are enough to inoculate the two media, this column gives the 

464 number of containers needed for both the media together. 

465 

10