## 2.2.7. Complex mixtures/UVCBs

Author: Pim N.H. Wassenaar

Reviewers: Joop de Knecht, Philipp Mayer

Learning objectives:

You should be able to

- Explain how complex mixtures/UVCBs differ from well-defined substances.
- Explain the challenges and uncertainties in the risk and hazard assessment of complex mixtures/UVCBs.

Key words: UVCB; Complex substances; Constituents

Introduction/type of substances

Beside substances that consist of a single chemical structure, there are also substances produced that contain multiple constituents each having its unique molecular structure. In general, three types of substances can be identified: 1) mono-constituent substances, 2) multi-constituent substances, and 3) UVCBs that are substances of Unknown or Variable composition, Complex reaction products or Biological materials (ECHA, 2017a). Mono-constituent substances contain one main constituent that makes up at least 80% of the substance (Figure 1A), whereas multi-constituent substances consist of several main constituents that are present at a concentration between 10% and 80% (Figure 1B). Potential other constituents within these substances are considered impurities (Figure 1A&B). These first two substance categories are sometimes also described as well-defined substances, as the composition is (or can be) well characterized. However, this section will specifically focus on the third category, the UVCB substances. UVCBs contain many different constituents of which some can be (partially) unknown and/or the exact composition can be variable or difficult to predict (Figure 1C). Principally, none of the constituents in a UVCB are considered as impurities. Although different terms are used to define UVCBs / complex chemical substances within various regulatory frameworks (Salvito et al., 2020), the term ‘UVCB’ will be used throughout this section to represent these various denominations.

Figure 1. Overview of the three main types of substances. A) mono-constituent substance. B) multi-constituent substance. C) UVCB substance. Pictures are derived from ECHA What is a substance? - ECHA (europa.eu).

Types and naming of UVCB substances

Several types of UVCB substances can be defined, including UVCBs that are synthesized or derived/refined from biological or mineral sources (ECHA, 2017a). Common types of UVCBs include:

- Extracts from plant or animal products (e.g. ‘Lavender, Lavandula hybrida, ext.’ [CAS 91722-69-9; EC 294-470-6]).
- Reaction products that are formed during a chemical reaction (e.g. ‘Reaction product of 1,3,4-thiadiazolidine-2,5-dithione, formaldehyde and phenol, heptyl derivs.’ [EC 939-460-0]).
- Products that are derived from industrial processes (e.g. ‘Naphtha (petroleum), catalytic reformed’ [CAS 68955-35-1; EC 273-271-8]).

In general, the name of a UVCB substance is a combination of its source (e.g. name of the species for biological sources, or name of the starting material for non-biological sources) and the used process(es) (e.g. extraction, fractionation, etc.) (ECHA, 2017a). In addition, for some UVCB categories, specific nomenclature systems are developed that can also include a description of the general composition or characteristics (e.g. physicochemical properties, like boiling range). A specific nomenclature system is for instance developed for hydrocarbon solvents and oleochemicals, in which the nomenclature is based on the chemical composition (e.g. ‘Hydrocarbons, C9-C11, n-alkanes, isoalkanes, cyclics, < 2% aromatics [CAS 64742-48-9; EC 919-857-5]’) (OECD, 2014; OECD, 2015).

Challenges in the risk and hazard evaluation of UVCBs

It is difficult to fully characterize the chemical composition of UVCBs as they can contain a relative large number of constituents. Generally, it is technically challenging or impossible to identify, and thus to test, all individual constituents present in a UVCB. As a consequence, a significant fraction is often defined as ‘unknown’ or is only specified in general terms (ECHA, 2017a,b). Nevertheless, specific information down to the individual constituent level could be relevant for risk/hazard assessment as some constituents can already cause effects at low concentrations (see chapter 6). In addition to an ‘unknown’ fraction, UVCBs can also have a variable composition. The composition may for instance depend on fluctuations in the manufacturing processes and/or source material, including spatial and temporal variations. Although this variability may not affect the functionality of the UVCB substance, it could influence and warrant a new hazard assessment (ECHA, 2017a,b; Salvito et al., 2020). Obviously, these key characteristics of UVCBs (i.e. the compositional complexity), complicate their risk and hazard assessment.

As it is not possible to identify, isolate and assess all individual constituents, alternative assessment approaches are being developed to evaluate UVCBs, including whole-substance and constituent based approaches. Within whole-substance based approaches, the UVCB is used as test-item. Testing of the whole substance might be relevant when the UVCB consists of structurally very similar constituents that are expected to have comparable fate and effect properties (Figure 2). However, when the UVCB displays a wide range of physicochemical properties a constituent based approach is generally preferred for risk/hazard assessment purposes, as the results of whole substance testing can be very difficult to interpret. For instance, the results of whole substance testing typically provide a single profile for the whole UVCB, while the fate, behavior and effects between (groups of) constituents could differ significantly. Furthermore, result interpretation might be challenging, as it could be difficult to maintain stable dosing/exposure concentrations when constituents with varying physicochemical properties are combined (e.g. due to differences in sorption, evaporation, solubility etc.).

Within constituent-based approaches, generally one or a few representative constituents are selected and evaluated. The results for these constituents are subsequently extrapolated to the other constituents, and ultimately to the UVCB. The selection of representative constituents can be based on several aspects, including in silico predictions of fate and hazard properties, the relevance or availability of the constituents and the structural variability (ECHA, 2017b; Salvito et al., 2020). To support the generation and selection of representative constituents computational methodologies are being developed (Dimitrov et al., 2015; Kutsarova et al., 2019).

One of the best described constituent based approaches is the ‘fraction profiling approach’, which is also known as the hydrocarbon block method (ECHA, 2017b; King, 1996). This method is specifically developed for petroleum substances (although it may also be applied to other UVCBs) and is applied in several risk assessment and PBT (Persistent, Bioaccumulative, Toxicity) assessment approaches (CONCAWE, 2016; Wassenaar and Verbruggen, 2021). Within the hydrocarbon block method, the composition of a UVCB is conceptually divided in blocks/fractions of (structurally) similar constituents. The underlying assumption is that all constituents within a block have fairly similar properties and could be assessed as if it is ‘one constituent’. More details on the hydrocarbon block method are provided in section 2.3.5 Hydrocarbons.

In general, the choice of the assessment approach is dependent on the substance and may also be dependent on the data already available as well as the stage and the general purpose of the assessment (e.g. PBT-assessment, risk assessment, etc.). In some cases, a combination of varying approaches could be considered most efficient. For instance by using a whole substance as test item in combination with analytical measurements of individual constituents over time.

Figure 2. UVCB substances X and Y contain constituents with varying physicochemical properties (colors). For substance X a whole-substance based approach might be used, whereas for substance Y a constituent based approach is generally preferred. The different shapes represent constituents that could potentially be grouped according to other properties, such as mode of toxic action. The ‘?’ represents an ‘unknown’ fraction. This figure is adopted and modified from Salvito et al. (2020).

References/useful links:

CONCAWE. (2016). PETRORISK Version 7.0 Public (Manual). https://www.concawe.eu/reach/petrorisk/.

Dimitrov, S., Georgieva, D.G., Pavlov, T.S., Karakolev, Y.H., Karamertzanis, P.G., Rasenberg, M., Mekenyan, O.G. (2015). UVCB substances: methodology for structural description and application to fate and hazard assessment. Environmental Toxicology and Chemistry 11, 2450-2462. https://doi.org/10.1002/etc.3100.

ECHA (2021). What is a substance. https://echa.europa.eu/support/substance-identification/what-is-a-substance

ECHA (2017a). Guidance for identification and naming of substances under REACH and CLP. European Chemicals Agency, Helsinki.

ECHA (2017b). Guidance on Information Requirements and Chemical Safety Assessment. Chapter R.11: PBT/vPvB assessment. European Chemicals Agency, Helsinki.

King, D.J., Lyne, R.L., Girling, A., Peterson, D.R., Stephenson, R., Short, D. (1996). Environmental risk assessment of petroleum substances: the hydrocarbon block method. CONCAWE report no. 96/52. Brussels.

Kutsarova, S.S., Yordanova, D.G., Karakolev, Y.H., Stoeva, S., Comber, M., Hughes, C.B., Vaiopoulou, E., Dimitrov, S.D., Mekenyan, O.G. (2019). UVCB substances II: Development of an endpoint-nonspecific procedure for selection of computationally generated representative constituents. Environmental Toxicology and Chemistry 38, 682-694. https://doi.org/10.1002/etc.4358.

OECD. (2015). OECD guidance for characterising hydrocarbon solvents for assessment purposes. Series on Testing and Assessment, No. 230. ENV/JM/MONO (2015)52. Organization for Economic Cooperation and Development, Paris.

OECD. (2014). OECD guidance for characterising oleochemical substances for assessment purposes. Series on Testing and Assessment, No. 193. ENV/JM/MONO(2014)6. Organization for Economic Cooperation and Development, Paris.

Salvito, D., Fernandez, M., Jenner, K., Lyon, D.Y., De Knecht, J., Mayer, P., MacLeod, M., Eisenreich, K., Leonards, P., Cesnaitis, R., León-Paumen, M., Embry, M., Déglin, S.E. (2020). Improving the environmental risk assessment of substances of unknown or variable composition, complex reaction products, or biological materials. Environmental Toxicology and Chemistry 39, 2097-2108. https://doi.org/10.1002/etc.4846.

Wassenaar, P.N.H., Verbruggen, E.M.J. (2021). Persistence, bioaccumulation and toxicity-assessment of petroleum UVCBs: A case study on alkylated three-ring PAHs. Chemosphere 276, 130113. https://doi.org/10.1016/j.chemosphere.2021.130113.