enzyme reach consortium guidance: how to populate iuclid 5 ... · in this example, einecs entries...

ERC/13/03 – ERC Guidance – updated OCT2013

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Enzyme REACH Consortium Guidance: How to populate IUCLID 5 Section 1 and test material information in Section 4 - 7 for enzymes

Disclaimer This communication is meant as guidance only. It is published by Enzyme REACH Consortium (ERC) in order to assist its members in their efforts to understand and comply with REACH. Please be reminded, however, that the REACH is the only authoritative legal text and that the present document does not substitute legal or otherwise expert advice. ERC and its members do not accept any liability for use of this communication or for activities contemplated and carried out under or relying on this communication.

Introduction The purpose of the guidance is to give practical information to Enzyme REACH Consortium (ERC)

members based on our experiences with 2010 and 2013 REACH dossiers. The guidance focuses on

specific descriptions on enzyme substances which may not be covered by ECHA’s support

documents.

ECHA has specifically addressed the issue of identification of enzyme substances, cf. section 4.3.2.3 of ECHA’s Guidance on Substance Identification1. IUCLID should be populated according to the identification. The relevant paragraphs of this guidance are inserted below. This guidance does not cover issues related to split and merge SIEFs. The enzyme substance should be regarded as a ‘UVCB1-substance’ due to its variability and partly unknown composition. … Enzyme substances are identified by the enzyme protein (IUBMB nomenclature) and the other constituents from the fermentation. … The enzyme substance typically contains 10-80 % (w/w) of the enzyme protein. The other constituents vary in percentage and depend on the production organism used, the fermentation medium, and operational parameters of the fermentation process as well as the downstream purification applied, but the composition will typically be within the ranges indicated in the following table.

Active enzyme protein 10 - 80%

Other proteins + peptides and amino acids

5 - 55%

Carbohydrates 3 - 40%

Lipids 0 - 5%

Inorganic salts 1 - 45%

Total 100 %

1 Guidance for identification and naming of substances under REACH and CLP. Reference: ECHA-11-G-10.1-

EN. Version 1.2, March 2012


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How to read the guidance The guidance text is black. Proposed text populated in IUCLID is blue Text which is variable depending on enzyme is orange.

Section 1. General Information

Section 1.1 Identification - Reference substance Reference substance for the enzyme should be selected. Reference substance may already exist in list of EINECS substances which are downloadable from IUCLID homepage (http://iuclid.eu/). There is certain information which should be manually populated.

General information

Information according to EINECS should be populated. Example is alpha-amylase.

Reference Substance Information

IUPAC Name

Populate IUBMB name (recommended name) and IUBMB number. This information is the primary identifier for enzymes under REACH.

Description

ERC recommend to populate the below text documenting enzyme identity and sameness of the enzyme according to ERC document “Safety evaluation of technical enzyme products with regards to the REACH legislation” ERpC/09/06. Text in color is specific to enzymes according to IUBMB identification. The example is alpha-amylase. According to the REACH Guidance on Substance Identification (Section 4.3.2.3), enzyme substances are identified by: 1) the catalytic activity of the enzyme protein (IUBMB nomenclature) and 2) the other constituents from the fermentation.

http://iuclid.eu/


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1) IUBMB nomenclature according to IUBMB (INTERNATIONAL UNION OF BIOCHEMISTRY AND MOLECULAR BIOLOGY (http://www.chem.qmul.ac.uk/iubmb/)). IUBMB name: alpha-amylase Enzyme Class No.: 3.2.1.1 Systematic name: 4-alpha-D-glucan glucanohydrolase Reaction: Acts on starch, glycogen and related polysaccharides and oligosaccharides in a random manner; reducing groups are liberated in the alpha-configuration. The term "alpha" relates to the initial anomeric configuration of the free sugar group released and not to the configuration of the linkage hydrolysed. 2) Constituents other than the enzyme protein from the fermentation The enzyme is produced by organisms which meet the criteria for “Safe Strain Lineage Concept" in "Safety evaluation of technical enzyme products with regards to the REACH legislation" dated March 25, 2009, published by Enzyme REACH Consortium (http://www.enzymes-reach.org/documents.html)

Synonyms

In this example, EINECS entries which are identical to alpha-amylase are populated. You may consider to write other known synonyms according to Enzyme Nomenclature database (http://www.chem.qmul.ac.uk/iubmb/enzyme/)

Related CAS number

If available, you may write CAS numbers which are linked to alpha-amylase (in the present example).

http://www.enzymes-reach.org/

http://www.enzymes-reach.org/

http://www.chem.qmul.ac.uk/iubmb/enzyme/


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Molecular and structural information

Molecular formula

Populate as follows. Not available. See Remarks.

Molecular weight range

Populate this section based on available scientific data.

Remarks

ERC recommends to populate the field with the following text. The substance is a protein; therefore the molecular formula is not available. The molecular weight varies according to origin and/or processing during secretion etc.


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Section 1.2 Substance Composition Several ERC policy documents argue that enzyme sameness is only dependent on the enzyme protein, not the constituents. Nevertheless, this section should be populated in order to pass the Technical Compliance Check. ERC recommends populating this section according to ECHA’s guidance on substance identification Section 4.3.2.3 (page 1 of this guidance).

Constituent (Reference substance should be created accordingly)

Range Typical concentration

Active enzyme protein of the enzyme

10 - 80% 50 %


5 - 55%

30%

Carbohydrates 3 - 40% 10 %

Lipids 0 - 5% 1 %

Inorganic salts 1 - 45% 9 %

Total 100 % 100 %

Please see Remarks for each component below.

Active enzyme protein of the enzyme

Remarks: In accordance with “GUIDANCE FOR SUBSTANCE IDENTIFICATION AND NAMING IN REACH Section 4.3.2.3”, the enzyme substance consists of 1) the active enzyme protein and 2) constituents other than the active enzyme protein. 1) Enzyme substance: The enzyme substance is identified according to catalytic activity defined by IUBMB (INTERNATIONAL UNION OF BIOCHEMISTRY AND MOLECULAR BIOLOGY (http://www.chem.qmul.ac.uk/iubmb/)). IUBMB name: alpha-amylase; Enzyme Class No.: 3.2.1.1; Reaction: Endohydrolysis of (1→4)-α-D-glucosidic linkages in polysaccharides containing three or more (1→4)-α-linked D-glucose units 2) Constituents other than enzyme protein. The substance does not contain a constituent which is ≥ 10 % (w/w) or relevant for classification and labelling and-or PBT assessment. The enzyme

http://www.chem.qmul.ac.uk/iubmb/)


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substance typically contains 10-80 % (w/w) of the enzyme protein. The other constituents vary in percentage and depend on the production organism used, the fermentation medium, and operational parameters of the fermentation process as well as the downstream purification applied, but the composition will typically be within the following ranges: Active enzyme protein 10 - 80%, Other proteins plus peptides and amino acids 5 - 55%, Carbohydrates 3 - 40%, Lipids 0 - 5%, Inorganic salts 1 - 45%. The enzyme substance is produced by organisms which meet the criteria for “Safe Strain Lineage Concept" in "Safety evaluation of technical enzyme products with regards to the REACH legislation" dated March 25, 2009, published by Enzyme REACH Consortium (http://www.enzymes-reach.org/). The constituents other than enzyme protein produced by the organisms meeting the above criteria do not contribute to classification, thereby the enzyme substance having the same catalytic activity from such safe organisms are considered as the same substance.


Description: It is a chemical group as a constituent of enzyme substance derived from the fermentation or extraction process (section 4.3.2.3 Guidance for Substance Identification and Naming in REACH). It consists of various proteins and peptides.

Carbohydrates

Description: It is a chemical group as a constituent of enzyme substance derived from the

fermentation or extraction process (section 4.3.2.3 Guidance for Substance Identification and

Naming in REACH). It consists of various carbohydrates.

Lipids

Description: It is a chemical group as a constituent of enzyme substance derived from the

fermentation or extraction process (section 4.3.2.3 Guidance for Substance Identification and

Naming in REACH). It consists of various lipids.

Inorganic salts

Description: It is a chemical group as a constituent of enzyme substance derived from the fermentation or extraction process (section 4.3.2.3 Guidance for Substance Identification and Naming in REACH). It consists of various inorganic salts.


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Section 1.4 Analytical Information ERC recommend that assay method(s) for each constituent is populated. Spectra data are not relevant for active enzyme protein and other constituents.

Constituent (Reference substance should be created accordingly)

Analytical method

Active enzyme protein of the enzyme Enzyme activity assay

Other proteins plus peptides and amino acids Any method e.g. Kjeldahl to determine protein, peptides and amino acid

Carbohydrates Any method e.g. colorimetric assay to determine carbohydrates or calculation

Lipids Any method to determine lipids

Inorganic salts Determination of ash

In each assay, a method should be uploaded and results should be populated.


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Test materials at each endpoint Information on the test material should be given in relation to each endpoint in sections 4-7. This is important information to certify that 1) the test material is representative for the enzyme identified in Section 1 and that 2) the presented data are reliable and based on investigation of a well characterized test material according to ERC safety policy. It is therefore crucial to indicate active enzyme protein in the test material as well as other constituents. There should be solid data to indicate stability of enzyme test materials under the specific test conditions of the individual endpoint study, since enzymes tend to lose activities over time depending on the conditions. Example: In case a repeated dose toxicity study has been performed, and the dosing solutions were prepared once a week during the study and stored at 4 ⁰C, it has to be documented that the enzyme is stable and therefore active at least for one week at 4 ⁰C, in the actual dilutions. Details on test material Notice that this section will be disseminated. Confidential information should be populated under the next section ”Confidential details on test material”. - Substance type: UVCB - Physical state: liquid - Stability under test conditions: <When relevant for the test conditions of the specific endpoint study, this section should be populated with stability data based on enzyme activity carried out by the data owner.> Example based on data: The test material is stable for at least 24 hours at room temperature and at 4 degrees of Celcius - Storage condition of test material: <This section should be based on stability test based on enzyme activity carried out by the data owner.> Example based on data: less than minus 18 degrees of Celsius Confidential details on test material <example> Confidential information on test batch can be populated under this section. - Name of test material (as cited in study report): Date, Enzyme name, batch number - Composition of test material, percentage of components: <Populate results as much as possible according to e.g. the analytical methods in Section 1.4. e.g. enzyme activity and amount of other


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constituents, Total organic solids (TOS) (i.e. 100% -(water + ash)) may be populated here, if available.> - Composition test date: - Lot/batch No.: XXXYYY - Expiration date of the lot/batch: Date


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Questions & Answers

General Question: Key question: Does this Guidance and indicated format replace the CEFIC SIP format that is used for chemicals? Answer: CEFIC SIP is a tool to facilitate sameness which is focused on well-defined substances. The ERC guidance does not facilitate sameness of an enzyme substance. It provides practical information on what to be written in relevant parts of IUCLID 5 on an enzyme substance. Description Question: Clear, this is the current CAS stated in IUBMB. What if there isn’t one, so just IUBMB? (mainly for ‘non phase-in’ enzymes) Answer: CAS is not required in REACH registration. You can leave it blank. Related CAS number Question: Here is not meant the read-across information? Just splits or deletions for confusing ID’s or duplicates from the past? Answer: We populated CAS number used for alpha-amylase by searching CAR Registry Number database. In Synonyms (above Related CAS number), we entered EINCES entries which should be merged into the alpha-amylase SIEF in order to make basis for merging other SIEFs in future. There are no fields for read-across. Read-across documentation was made under “endpoint” in IUCLID Section 4 – 7. Molecular weight range Question: Is meant here that enzyme maybe active as monomer or dimer? Makes calc of % variable& difficult. Answer: We did not particularly mention monomer or dimers, but molecular variations in general – from different origins and processing of protein at secretion. It can be the same of monomer or dimers, which I am not so familiar with … Catalase, for example?? But this description should cover such a case, too. Section 1.2 Substance Composition Question: Have the ranges been kept the same for registrations this far or do they vary with the (split) SIEFs? Characterise this for each Enzyme (category) based on shared info in the SIEF / group (see EINECS-IUBMB list). How does this work for lower percentages. ≥10% rule for identification of constituents does not work here? Answer: We propose the same value for all enzymes. 10% rule is not relevant for enzymes since enzymes have their own identification section as specific UVCB in ECHA’s guidance. Active enzyme protein of the enzyme under Constituents Question: This reference substance ID differs per SIEF. What meaning will be assigned, when ECHA assigns a new (‘mono-constituent’) EU number to this? Answer: This is a constituent as active enzyme protein. As defined by ECHA, an enzyme substance normally consists of multi-constituents. This guidance covers this situation. If an enzyme substance contains more than 80 % active enzyme protein, it is possible to make an approach to register the enzyme as mono-component substance. Then any other substances contained in the enzyme substance shall be handled as impurities. Constituents Question: Why do we document constituents other than enzyme protein in active enzyme protein under “Active enzyme protein”? Answer: Reference substance used in Section 1.1 originates from the EINECS inventory Question: Can we share the reference substances from here since they are supposed to be generic? Answer: Yes, we will share the IUCLID reference substance files.


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Section 1.4 Analytical Information Question: e.g. Can we share advisable or typical analytical methods? Answer: We recommend well-established assay methods for each constituent. Confidential details on test material - Composition of test material, percentage of components Questions/comment: Likely is that this composition cannot be broken down as in sec. 1.2? Answer: Noted. It is our experience that the amount of active enzyme protein is at least critical to document in this section so that we can assess data based on active enzyme protein.

enzyme reach consortium guidance: how to populate iuclid 5 ... · in this example, einecs entries...

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