SC I C. Bacterial Endotoxin Testing

This section provides an exposition of the Commission’s policy and information on its implementation. The guidelines of the European Pharmacopoeia are included as an Annex.

The test for bacterial endotoxins of the European Pharmacopoeia (Ph Eur) is included as Appendix XIV C of the British Pharmacopoeia. This text has been prepared in collaboration with the Japanese Pharmacopoeia and the United States Pharmacopeia.

1. This in vitro test is being progressively applied in appropriate monographs of both the British and European Pharmacopoeia in place of the in vivo test for pyrogens.

2. Methods for the detection of Gram-negative bacterial endotoxins are based on the use of a lysate of amoebocytes from the horseshoe crab (Limulus polyphemus or Tachypleus tridentatus). Addition of endotoxin to this lysate may result in gelation, precipitation or turbidity. The method used in the monographs of the European and British Pharmacopoeias is, unless otherwise stated, that using a gelation end-point.

3. A European Pharmacopoeia Biological Reference Preparation (BRP) of Endotoxin calibrated in International Units (IU) has been established for use in this test. The current BRP (batch 5) was established following an international collaborative study. It consists of endotoxin from the same bulk as the Third International Standard established by the World Health Organization and the current standard established by the Food and Drug Administration and the United States Pharmacopeia for use in the United States of America (EC-7). Following adoption of the recommendations in the report of the collaborative study¹ global harmonisation of endotoxin unitage has been maintained², that is, the FDA/USP Endotoxin Unit (EU) is equivalent to the International Unit (IU).

General policy

4. In any individual monograph only one test is required, either that for pyrogens or that for bacterial endotoxins.

5. In the absence of evidence to the contrary, the test for bacterial endotoxins is preferred, since it is considered usually to provide equal or better protection to the patient.

6. Before including a test for bacterial endotoxins in a monograph, evidence is required that a test, as described in Appendix XIV C, can be applied satisfactorily to the item in question.

7. The necessary information is sought from manufacturers. Companies are invited to provide any validation data that they have concerning the applicability of the test for bacterial endotoxins to the substances and formulations of interest.

7.1 Such data should include details of sample preparation and of any procedures necessary to eliminate interfering factors.

7.2 In addition, any available parallel data for rabbit pyrogen testing that would contribute to an assurance that the replacement of a rabbit pyrogen test by the test for bacterial endotoxin is appropriate, should be provided.

7.3 For formulated preparations, a distinction should be made between any sample treatment necessitated by excipients included in a particular product and any required due to the nature of the active constituent. Attention should also be drawn to any manipulation normally required for the active constituent that is rendered redundant by the composition of the formulation, for example, pH adjustment.

8. In order to set an appropriate limit for bacterial endotoxins it is necessary to know the intended route of parenteral administration (in particular, if the substance may be administered intrathecally) together with the maximum dose as recommended in relevant product data sheets. The limit for a given material or preparation is expressed as the endotoxin limit (EL) or endotoxin limit concentration (EL). The EL may be expressed in IU of endotoxin per millilitre for a defined solution of the material or preparation, or, for some medicinal substances, in IU of endotoxin in relation to a defined quantity of the material (that is, per milligram or, for biologically assayed materials, per IU) of material. The limit, which is stated in the monograph, is usually established on the following basis:

EL = K/M

8.1 K, sometimes referred to as the minimum pyrogenic dose, is the maximum number of IU of endotoxin which the patient may receive without suffering toxic reactions. The appropriate value for K will be taken from the table below.

8.2 M is the maximum dose of the drug substance per person (or per kg) per hour. This is interpreted as the maximum amount that might be administered within one hour. For subcutaneous (SC), intramuscular (IM) or bolus intravenous (IV) injections this will be an entire single dose. For intravenous infusions given over a prolonged period it is the proportion of the dose that would be infused during one hour and depends upon the rate of infusion. The value used is the maximum dose recommended by the manufacturer and stated in the relevant product data sheet. It is accepted that in exceptional circumstances this dose may be exceeded at the discretion of the physician; such use is outside the scope of the Pharmacopoeia.

Implementation

9. The British Pharmacopoeia Commission is seeking to replace the test for pyrogens by that for bacterial endotoxins wherever possible in the Pharmacopoeia. The European Pharmacopoeia Commission has a similar policy and has indicated that, for monographs already published, the change will be carried out, where appropriate, whenever the monograph in question is revised.

9.1 The test for bacterial endotoxins is now specified in the European Pharmacopoeia monograph for Water for Injections and in many other monographs including a range of antibiotics (for example, Doxorubicin Hydrochloride and Oxytetracycline Hydrochloride), biological materials (for example, Somatropin and Heparin) and radiopharmaceutical preparations.

9.2 Revision of the European Pharmacopoeia monograph for Parenteral Preparations to permit the use of the test for bacterial endotoxins in defined circumstances opened the way for the test for pyrogens to be replaced by that for bacterial endotoxins in individual monographs for parenteral preparations in the British Pharmacopoeia. The necessary change has already been made in a wide range of monographs including those for certain biological formulations such as Protamine Sulfate Injection and in the monographs for a number of widely used intravenous infusions such as Sodium Chloride Intravenous Infusion. Appropriate data are being sought from manufacturers for the remaining monographs for which a test for pyrogens is specified.

Annex

Guidelines concerning the test for Bacterial endotoxins have been published as an Annex to the method text 2.6.14 in the European Pharmacopoeia and are reproduced here.

The following section is published for information.

¹ Poole, S., Dawson, P., Gaines Das, R.E. (1997). Second international standard for endotoxin: calibration in an international collaborative study. Journal of Endotoxin Research 4 (3), 221–231.

² Poole, S. et al. (2012). WHO international standard for endotoxin: report of an international collaborative study to evaluate three preparations of endotoxin for their suitability to serve as the third international standard for bacterial endotoxin. World Health Organisation.