INTERNATIONAL
`STANDARD
`
`ISO
`10993-7
`
`Second edition
`2008-10-15
`
`Biological evaluation of medical
`devices-
`Part 7:
`Ethylene oxide sterilization residuals
`
`Evaluation biologique des dispositifs medicaux -
`Parlie 7: Residus de sterilisation a l'oxyde d'ethylene
`
`C0P'Y'/'¥1ht International OrQaniz.atlon for Slandardizatlon
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`
`NOi for Resale
`
`Reference number
`ISO 10993-72008(E)
`
`© ISO2008
`
`Novartis Exhibit 2196.001
`Regeneron v. Novartis, IPR2021-00816
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`

`

`ISO 10993-7:2008(E)
`
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`

`ISO 10993-7:2008(E)
`
`Contents
`
`Page
`
`Foreword............................................................................................................................................................ iv
`Introduction ....................................................................................................................................................... vi
`1
`Scope ......................................................................................................................................................1
`2
`Normative references ............................................................................................................................1
`3
`Terms and definitions ...........................................................................................................................1
`4
`Requirements .........................................................................................................................................2
`4.1
`General....................................................................................................................................................2
`4.2
`Categorization of devices .....................................................................................................................2
`4.3
`Allowable limits......................................................................................................................................3
`4.4
`Determination of EO and ECH residuals .............................................................................................5
`5
`Product release ....................................................................................................................................10
`5.1
`General..................................................................................................................................................10
`5.2
`Release of products without dissipation curve data .......................................................................10
`5.3
`Procedure for product release using residue dissipation curves ..................................................10
`Annex A (normative) Evaluation of gas chromatograms..............................................................................12
`Annex B (informative) Gas chromatographic determination for EO and ECH............................................15
`Annex C (informative) Flowchart and guidance for the application of this part of ISO 10993 series
`of standards to the determination of EO and ECH residuals in medical devices.........................19
`Annex D (informative) Factors influencing product residual........................................................................26
`Annex E (informative) Extraction conditions for determination of residual EO .........................................28
`Annex F (informative) Rationale for the provisions of this part of ISO 10993 ............................................29
`Annex G (informative) Establishment of allowable limits for EO .................................................................33
`Annex H (informative) Establishment of allowable limits for ECH...............................................................50
`Annex I (informative) Establishment of allowable limits for EG ...................................................................59
`Annex J (informative) Preparation of EO and ECH standards......................................................................63
`Annex K (informative) Ethylene oxide residue measuring methods ...........................................................67
`Bibliography ......................................................................................................................................................74
`
`
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`

`ISO 10993-7:2008(E)
`
`Foreword
`
`ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies
`(ISO member bodies). The work of preparing International Standards is normally carried out through ISO
`technical committees. Each member body interested in a subject for which a technical committee has been
`established has the right to be represented on that committee. International organizations, governmental and
`non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the
`International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
`
`International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
`
`The main task of technical committees is to prepare International Standards. Draft International Standards
`adopted by the technical committees are circulated to the member bodies for voting. Publication as an
`International Standard requires approval by at least 75 % of the member bodies casting a vote.
`
`Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
`rights. ISO shall not be held responsible for identifying any or all such patent rights.
`
`ISO 10993-7 was prepared by Technical Committee ISO/TC 194, Biological evaluation of medical devices.
`
`This second edition cancels and replaces the first edition (ISO 10993-7:1995) which has been technically
`revised.
`
`ISO 10993 consists of the following parts, under the general title Biological evaluation of medical devices:
`
` Part 1: Evaluation and testing within a risk management system
`
` Part 2: Animal welfare requirements
`
` Part 3: Tests for genotoxicity, carcinogenicity and reproductive toxicity
`
` Part 4: Selection of tests for interactions with blood
`
` Part 5: Tests for in vitro cytotoxicity
`
` Part 6: Tests for local effects after implantation
`
` Part 7: Ethylene oxide sterilization residuals
`
` Part 9: Framework for identification and quantification of potential degradation products
`
` Part 10: Tests for irritation and skin sensitization
`
` Part 11: Tests for systemic toxicity
`
` Part 12: Sample preparation and reference materials
`
` Part 13: Identification and quantification of degradation products from polymeric medical devices
`
` Part 14: Identification and quantification of degradation products from ceramics
`
` Part 15: Identification and quantification of degradation products from metals and alloys
`
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`

`ISO 10993-7:2008(E)
`
` Part 16: Toxicokinetic study design for degradation products and leachables
`
` Part 17: Establishment of allowable limits for leachable substances
`
` Part 18: Chemical characterization of materials
`
` Part 19: Physico-chemical, morphological and topographical characterization of materials [Technical
`Specification]
`
` Part 20: Principles and methods
`Specification]
`
`for
`
`immunotoxicology
`
`testing of medical devices
`
`[Technical
`
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`

`ISO 10993-7:2008(E)
`
`Introduction
`
`Requirements for the development, validation and routine control of an ethylene oxide sterilization process for
`medical devices are given in International Standards developed by ISO/TC 198. Certain requirements relating
`to medical devices for biological testing, selection of tests, and the allocation of devices to categories are dealt
`with in a variety of International Standards developed by ISO/TC 194. The specific requirement for ethylene
`oxide and other sterilization process residuals was referred to ISO/TC 194. Other International Standards
`delineate particular requirements for biological testing for specific products.
`
`As noted in the introduction to ISO 11135-1:2007, when determining the suitability of ethylene oxide (EO) for
`sterilization of medical devices, it is important to ensure that the levels of residual EO, ethylene chlorohydrin
`(ECH) and ethylene glycol (EG) pose a minimal risk to the patient in normal product use. Therefore, it is
`important that the use of alternative materials and sterilization processes be considered during product design
`and development. EO is known to exhibit a number of biological effects. In the development of this part of
`ISO 10993, consideration was given to these effects, which include irritation, organ damage, mutagenicity and
`carcinogenicity in humans and animals, and reproductive effects in animals. Similar consideration was given
`to the harmful effects of ECH and EG. In practice, for most devices, exposure to EO and ECH is considerably
`lower than the maximum values specified in this part of ISO 10993.
`
`Moreover, when the choice for EO sterilization has been made, irrespective of the provisions of this part of
`ISO 10993, exposure to EO residues should be minimized. Requirements herein are in addition to the
`biological evaluation and testing requirements for each individually designed medical device as indicated in
`ISO 10993-1. The biological evaluation and testing requirements, combined with the EO-sterilization process
`residue limits, form the justification that an EO-sterilized device is acceptable for use. Maximum allowable
`residues for ethylene chlorohydrin (ECH), when ECH has been found to be present in medical devices
`sterilized with EO, are also specified. Local effects (e.g., irritation) have been considered and are incorporated
`in the tolerable contact limit (TCL) as given in 4.3.5.2 and Annex G for EO, and in 4.3.5.3 and Annex H for
`ECH.
`
`
`
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`

`

`INTERNATIONAL STANDARD
`
`ISO 10993-7:2008(E)
`
`
`
`Biological evaluation of medical devices —
`
`Part 7:
`Ethylene oxide sterilization residuals
`
`1 Scope
`
`This part of ISO 10993 specifies allowable limits for residual ethylene oxide (EO) and ethylene chlorohydrin
`(ECH) in individual EO-sterilized medical devices, procedures for the measurement of EO and ECH, and
`methods for determining compliance so that devices may be released. Additional background, including
`guidance and a flowchart showing how this document is applied, are also included in the informative annexes.
`
`EO-sterilized devices that have no patient contact (e.g., in vitro diagnostic devices) are not covered by this
`part of ISO 10993.
`
`NOTE
`
`This part of ISO 10993 does not specify limits for ethylene glycol (EG).
`
`2 Normative references
`
`The following referenced documents are indispensable for the application of this document. For dated
`references, only the edition cited applies. For undated references, the latest edition of the referenced
`document (including any amendments) applies.
`
`ISO 10993-1:—1), Biological evaluation of medical devices — Part 1: Evaluation and testing within a risk
`management process
`
`ISO 10993-3, Biological evaluation of medical devices — Part 3: Tests for genotoxicity, carcinogenicity and
`reproductive toxicity
`
`ISO 10993-10, Biological evaluation of medical devices — Part 10: Tests for irritation and delayed-type
`hypersensitivity
`
`ISO 10993-12, Biological evaluation of medical devices — Part 12: Sample preparation and reference
`materials
`
`ISO 10993-17:2002, Biological evaluation of medical devices — Part 17: Establishment of allowable limits for
`leachable substances
`
`3 Terms and definitions
`
`For the purposes of this document, the terms and definitions given in ISO 10993-1, ISO 10993-17 and the
`following apply.
`
`3.1
`simulated-use extraction
`extraction to demonstrate compliance with the requirements of this part of ISO 10993, by evaluating residue
`levels available to the patient or user from devices during the routine use of a device with water extraction to
`simulate product use
`
`
`1) To be published. (Revision of ISO 10993-1:2003)
`
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`

`ISO 10993-7:2008(E)
`
`3.2
`exhaustive extraction
`extraction until the amount of EO or ECH in a subsequent extraction is less than 10 % of that detected in the
`first extraction, or until there is no analytically significant increase in the cumulative residue levels detected
`
`As it is not possible to demonstrate the exhaustive nature of residual recovery, the definition of exhaustive
`NOTE
`extraction adopted is as above.
`
`4 Requirements
`
`4.1 General
`
`Information on the derivation of the limits in this part of ISO 10993 as well as other important background
`NOTE
`information and guidance relevant to the use of this document is contained in the informative annexes.
`
`This clause specifies maximum allowable residues for ethylene oxide (EO) for each individual medical device
`sterilized with EO. As noted in the introduction to ISO 11135-1:2007, when determining the suitability of EO for
`sterilization of medical devices, it is important to ensure that the levels of residual EO, ethylene chlorohydrin
`(ECH) and ethylene glycol (EG) pose a minimal risk to the patient in normal product use. Moreover, when the
`choice for EO sterilization has been made, irrespective of the provisions of this standard, exposure to EO
`residues should be minimized. Maximum allowable residues for ECH, when ECH has been found to be
`present in medical devices sterilized with EO, are also specified. Local effects (e.g., irritation) have been
`considered and are incorporated in the tolerable contact limit (TCL) as discussed in 4.3.5.2 and Annex G for
`EO, and 4.3.5.3 and Annex H for ECH. No device limits are specified for EG because a risk assessment
`(Annex I) indicates that calculated allowable levels are higher than those likely to occur in a medical device.
`However, the potential exists for acute haemodynamic and haemolytic effects to occur following rapid
`intravenous administration of hyperosmolar compounds like EG. Ethylene oxide sterilization of medical
`devices would not be expected to produce hyperosmolar solutions. Methods for the determination of EO and
`ECH are given in 4.4.
`
`The requirements in this part of ISO 10993 are in addition to the biological testing requirements set out in
`ISO 10993-1. For devices sterilized using ethylene oxide, attention shall be paid in particular to ISO 10993-3
`and ISO 10993-10. All applicable requirements of ISO 10993-1 shall take into account the EO residual level at
`the time of release for each individually designed medical device.
`
`Results of the biological assessment of the device may dictate more stringent limits than those specified
`in 4.3, which are designed to protect against systemic effects.
`
`4.2 Categorization of devices
`
`In establishing the maximum daily doses of EO and ECH that a medical device is allowed to deliver to
`patients, devices shall be categorized according to the duration of contact.
`
`Devices shall be placed into one of three exposure categories in accordance with ISO 10993-1:—, 5.3:
`
`a)
`
`limited exposure (A) – devices whose cumulative single, multiple or repeated use or contact is up to 24 h;
`
`b) prolonged exposure (B) – devices whose cumulative single, multiple, or repeated long-term use or
`contact is likely to exceed 24 h but not 30 d;
`
`c) permanent contact (C) – devices whose cumulative single, multiple or repeated long-term use or contact
`exceeds 30 d.
`
`If a material or device can be placed in more than one duration category, the more rigorous testing and/or
`evaluation considerations should apply. With multiple exposures, the decision into which category a device is
`placed should take into account the potential cumulative effect, bearing in mind the period of time over which
`these exposures occur.
`
`As it is applied in this part of ISO 10993, “multiple use” is defined to mean repeated use of the same device
`NOTE
`type, e.g. dialyser cartridges.
`
`2
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`

`ISO 10993-7:2008(E)
`
`4.3 Allowable limits
`
`4.3.1 General
`
`For each medical device, the maximum allowable doses of EO and ECH delivered to patients shall not exceed
`the values given below for the exposure category that the device has been placed into in accordance with 4.2.
`
`The limits for permanent contact and prolonged exposure devices are expressed as maximum average daily
`doses. These limits carry additional constraints for the first 24 h of the exposure period and, in the case of the
`permanent contact devices, for the first 30 days. These constraints place limitations on the amount of EO and
`ECH that can be delivered to the patient during these early time periods. If data are available, consideration
`should be given for proportioning the limits downward if multiple devices with the residue of concern are used
`at one time, or proportioning the limits upward when device use is only for a part of the exposure period of
`concern. These concomitant exposure factors (CEF) and proportional exposure factors (PEF) are given in
`ISO 10993-17. The procedure that was used to establish the allowable limits is described in Annex G for EO,
`in Annex H for ECH, and the rationale for considering the establishment of allowable limits for EG is described
`in Annex I.
`
`4.3.2 Permanent contact devices
`
`The average daily dose of EO to patient shall not exceed 0,1 mg/d. In addition, the maximum EO dose shall
`not exceed:
`
` 4 mg in the first 24 h;
`
` 60 mg in the first 30 d;
`
` 2,5 g in a lifetime.
`
`The average daily dose of ECH to patient shall not exceed 0,4 mg/d. In addition, the maximum ECH dose
`shall not exceed:
`
` 9 mg in the first 24 h;
`
` 60 mg in the first 30 d;
`
` 10 g in a lifetime.
`
`4.3.3 Prolonged exposure devices
`
`The average daily dose of EO to patient shall not exceed 2 mg/d. In addition, the maximum EO dose shall not
`exceed:
`
` 4 mg in the first 24 h;
`
` 60 mg in the first 30 d.
`
`The average daily dose of ECH to patient shall not exceed 2 mg/d. In addition, the maximum ECH dose shall
`not exceed:
`
` 9 mg in the first 24 h;
`
` 60 mg in the first 30 d.
`
`4.3.4 Limited exposure devices
`
`The average daily dose of EO to patient shall not exceed 4 mg.
`
`The average daily dose of ECH to patient shall not exceed 9 mg.
`
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`

`ISO 10993-7:2008(E)
`
`4.3.5 Tolerable contact limits for surface contacting devices and implants
`
`4.3.5.1 Overview
`
`The tolerable contact limit (TCL) is expressed in units of micrograms per square centimetre for EO and
`milligrams per square centimetre for ECH. The unit of square centimetre represents the surface area of the
`patient-device interface.
`
`NOTE
`
`The intent of this subclause is to prevent localized irritation due to EO or ECH released from the device.
`
`4.3.5.2
`
`Tolerable contact limit for EO
`
`Either the EO TCL for surface contacting devices and implants shall not exceed 10 µg/cm2 or it shall exhibit
`negligible irritation as specified in ISO 10993-10.
`
`4.3.5.3
`
`Tolerable contact limit for ECH for surface contacting devices
`
`Either the ECH TCL for surface contacting devices and implants shall not exceed 5 mg/cm2 or it shall exhibit
`negligible irritation as specified in ISO 10993-10.
`
`4.3.6 Special situations
`
`For multi-device systems the limits shall apply to each individual patient-contact device.
`
`Residue of EO in intraocular lenses shall not exceed 0,5 µg EO per lens per day, or 1,25 µg per lens. Prorate
`limits for other intraocular devices are set on the basis of the mass of the device, with the mass of an
`intraocular lens taken as 20 mg. The acceptability of ECH levels in intraocular devices made from viscoelastic
`materials that contain chlorine may need to be evaluated, as the level of ECH that results in ocular toxicity is
`about four times greater than the corresponding EO level.
`
`For blood cell separators used in patient and donor blood collection, the maximum allowable dose of EO is
`10 mg and the maximum allowable dose of ECH shall not exceed 22 mg.
`
`For blood oxygenators and blood separators, the maximum allowable dose of EO to patient is 60 mg and the
`maximum allowable dose of ECH shall not exceed 45 mg.
`
`For devices used in cardiopulmonary bypass procedures, the maximum allowable limits shall be 20 mg for EO
`and 9 mg for ECH.
`
`For extracorporeal blood purification devices, the EO and ECH limits specified shall be 4,6 mg/device, but the
`allowable EO dose for a lifetime may be exceeded.
`
`For drapes that are intended to contact only intact skin, the maximum allowable limits shall be the TCL of
`10 g/cm2 for EO and 5 mg/cm2 for ECH, or the drapes shall exhibit negligible irritation as specified in
`ISO 10993-10.
`
`The rationale for specifying EO limits for certain devices that are at variance with the general requirements
`NOTE
`appears in Annex F.
`
`A flowchart providing guidance for the application of this part of ISO 10993 to the determination of EO
`residuals in medical devices is presented in Annex C.
`
`4
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`

`ISO 10993-7:2008(E)
`
`4.4 Determination of EO and ECH residuals
`
`4.4.1 General
`
`4.4.1.1
`
`Procedure
`
`The procedure for determining compliance with 4.3 consists of extracting the residue from samples,
`determining the amount of residue, determining the contact surface of the device, and analysing and
`interpreting the data.
`
`DANGER — Analysts and others who obtain samples should perform all work involving the use of the
`chemicals and solvents required for these methods in a fume cupboard whilst wearing appropriate
`protective clothing, and should review the Material Safety Data information for each chemical prior to
`such use. Healthcare workers using EO-sterilized medical devices shall take appropriate precautions
`to protect against exposure to residues, which may be required by local occupational health and
`safety regulations.
`
`4.4.1.2
`
`Ethylene oxide
`
`This is a flammable gas that is irritating to body surfaces and highly reactive. It is mutagenic under many
`conditions, has fetotoxic and teratogenic properties, can adversely effect testicular function and can produce
`injury to many organ systems in the body. In cancer studies in animals, inhalation exposure produced several
`types of neoplastic changes including leukaemia, brain tumours and mammary tumours while ingestion or
`subcutaneous administration produced tumours only at the site of contact. One investigator has reported
`higher cancer and mortality rates in some subpopulations of exposed workers. However, the results of several
`studies in workers have shown even weaker associations. See References [177], [178] and [181]. In 1994 the
`International Agency for Research on Cancer (IARC) reclassified EO as a human carcinogen (class 1) based
`mainly on its mechanism of action. See Reference [75].
`
`4.4.1.3
`
`Ethylene chlorohydrin
`
`This is a flammable liquid that is irritating to body surfaces, acutely toxic and readily absorbed through the skin
`in toxic amounts. It has weak mutagenic potential, has some potential to produce fetotoxic and teratogenic
`changes and can produce injury to several organ systems in the body including lungs, kidneys, central
`nervous system and cardiovascular system. It was negative in cancer bioassays in animals.
`
`4.4.2 Determination of residue
`
`A valid method of extraction and measurement shall be used to determine the amount of EO and, where
`necessary, ECH delivered to the patient.
`
`If ECH is not detected based on the results of analyses performed using the methods given in either K.4.2 or
`K.4.7, no further monitoring for ECH is required.
`
`Many gas chromatography (GC) methods that use a capillary column instead of a packed column will produce
`NOTE
`EO, ECH and EG results during a single sample run.
`
`The guiding principle in selecting appropriate extraction methods (4.4.6) for the quantitative determination of
`EO and, where necessary, ECH is the evaluation of the dose to the patient in order to show compliance with
`the requirements set out in 4.3.
`
`Where residues are shown to be within the requirements for products tested by exhaustive extraction, there is
`no need to further challenge the device by simulated-use extraction, provided all applicable limits in 4.3 are
`met. When exhaustive extraction is used, particular attention shall be paid to the limits expressed for the first
`24 h and for the first 30 days in 4.3.
`
`Many analytical methods for these EO-sterilization residuals have been described and are reviewed in the
`Bibliography. However, the enormous diversity of materials and methods of construction of sterile medical
`
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`

`

`ISO 10993-7:2008(E)
`
`devices may, in certain cases, still present problems in determining residual EO and ECH levels using the
`methods given in the Bibliography. Therefore, any method that has been shown to be analytically sound (i.e.
`demonstrated accuracy, precision, linearity, sensitivity, and selectivity) may be used, provided that it has been
`validated. Annex A contains general validation requirements for gas chromatographic methods.
`
`4.4.3 Product sampling and sample “blank”
`
`4.4.3.1
`
`Product sampling
`
`Samples to be used for residual analysis shall be selected in such a manner as to be truly representative of
`the product. When selecting samples, attention shall be given to the many factors described in Annex D.
`Since many of these factors influence not only the initial levels of residuals in device components but also the
`rate of residue dissipation, they shall also be considered when test samples are drawn from a processed load
`and sent to the laboratory for analysis. Removal of the product samples from the processed load soon after a
`sterilization cycle is completed and shipment to a laboratory far from the sterilization site or storage in the
`laboratory for later analysis can jeopardize correlations of residual levels on the samples with those on the
`rest of the load. Moreover, if samples cannot be drawn from the load and handled so that the effect on
`aeration conditions for the sample will be negligible, an experiment to establish the relationship between the
`sample aeration and load aeration at various seasons of the year shall be carried out.
`
`Precautions shall be taken to minimize or control the effects of laboratory conditions on the rate of aeration for
`test samples that have been removed from a product load (see D.1.5). In addition, operator and analyst safety
`shall be ensured. Samples should remain with the product load until the day of analysis or until test samples
`are retrieved and immediately frozen. The time between removal of samples from a controlled aeration area
`and the beginning of extraction should be held to a minimum. Samples shall be sealed, shipped and stored
`frozen when analysis is delayed. Samples shall be shipped on dry ice on overnight delivery. Dry ice shall
`remain in the shipping container throughout the shipment and be present when the package is opened in the
`laboratory. Test samples may also be taken directly from the product load at the desired aeration interval and
`immediately placed into a headspace vial, which is sealed and then shipped to the laboratory for analysis. As
`an alternative, samples may be extracted and the extraction fluid shipped to the analytical laboratory for
`analysis. If the extraction fluid is water, then shipment shall be done such that the fluid is kept at ice-cold
`temperatures (< 10 °C) until arrival. Testing should be carried out to measure hydrolysis of EO to EG.
`
`Samples to be analysed shall be placed in a fume cupboard and removed from the packaging. Samples shall
`be prepared according to any applicable pre-use instructions in the product labelling. Extractions shall be
`started as soon as possible after the device has been removed from the packaging or pre-use preparations
`have been completed.
`
`4.4.3.2
`
`Sample “blank”
`
`To ensure that no other sample matrix components with the same retention time as any of the residues being
`determined are present, a “blank” sample shall be evaluated for the possible presence of such interferences
`by the extraction of a non-sterilized sample using the identical procedure being applied to the EO-sterilized
`samples. In the event of materials being extracted from such a “blank” with conflicting or overlapping retention
`times in the GC analysis, chromato