How to answer basic physical chemical questions for your chemical and agrochemical substances when test guidance changes and as regulatory standards evolve

Although most physiochemical endpoints are standard for crop and chemical active substances, selection of the right test method to assess them is critical. This blog reviews some standard endpoints and looks at the tests used, in the light of changing regulatory expectations and revised test methodologies for difficult substances.

The evolving environment influencing physical chemical testing

Key physical chemistry endpoints have been well established for many years and little has changed in this area of crop and chemical science.  Physical chemistry testing is a critical foundational activity for all crop and chemical active substances and clear test guidelines exist for most endpoints.  However, accompanying test guidance and methodologies have changed over time reflecting improvements in instrumentation and more rigorous data requirements from regulators, driven initially by regulations such as EU REACH (Registration, Evaluation, Authorisation and restriction of CHemicals).  In addition, endpoints that were previously waived because the test methodology was unsuitable for a specific type of substance, e.g., partition coefficient for surface-active agents, can now be assessed with advances in methods. This may have implications for established chemicals and crop protection active substances that achieved approval based on older physical chemistry tests or data waivers.

Spotlight on some key physical chemical tests and their application

The following section focuses on three key endpoints where there have been changes in methodologies and testing approaches. The changes in methods have been driven by changes in regulatory requirements and this illustrates the importance of always considering your physical chemical endpoints within the current regulatory context.

Water solubility: using the critical micelle concentration (CMC) test for surfactants

Water solubility is a basic physical chemical parameter of a substance; but what if that substance is a surfactant? Surfactants, or surface-active agents, are designed to reduce the surface tension of liquids to allow better mixing of phases or increased wetting.

Standard tests for determining water solubility

The standard tests used for determining water solubility are OECD 105 and the equivalent EU A.6. These tests recommend the column elution or flask methods for determining water solubility but neither is applicable to surface-active agents because emulsions, which prevent accurate analysis, can be generated in the test system, as undissolved material that cannot be removed from saturated test solutions.

Rationale for using the CMC test to determine water solubility

The CMC is an important characteristic of a surfactant and is defined as the concentration of surfactants above which micelles form, and all additional surfactant forms micelles. As increasing concentrations of surfactant are added to water, the surface tension decreases until the minimum surface tension is reached and micelles start to form – this is the CMC. After this point, further increases in surfactant concentration have little impact on surface tension, which remains relatively constant, as all additional surfactant forms micelles. In terms of water solubility, below the CMC, the surfactant is considered to be thermodynamically soluble in the water while, above the CMC, the solubility of the surfactant in water is exceeded. Thus, the water solubility of a surfactant may be expressed in terms of the CMC.

Utilizing the CMC test in practice

The key measurement used for determining CMC is surface tension, measured using a tensiometer. Simple dilutions of a strong, fully dispersed stock solution of test substance are used and the surface tension of each dilution is measured. Surface tension is then plotted against concentration using a logarithmic scale. Linear regressions are fitted to the two parts of the graph and the point of intersection determines the CMC – see the example.

Figure 1: Surface tension versus log concentration of a surface-active substance

Implications for crop or chemical active substance registrations or reapprovals

Older chemicals may not have data for water solubility estimated in this way and this could be a gap within existing dossiers.

Partition coefficient: using OECD 123 for substances with POW >4

The partition coefficient is defined as the ratio of the equilibrium concentrations of a dissolved substance in a two-phase system consisting of two immiscible solvents. For testing purposes, this is usually the partition coefficient of the test substance between water and 1-octanol (POW). It is used to establish whether a substance tends to be hydrophobic or lipophilic and provides information about how the substance is likely to be distributed in animal tissues and the wider environment. The value obtained can indicate the bioaccumulation potential of the substance in the environment. However, not all tests are applicable for surface-active substances and, whilst, historically, regulators may have accepted the absence of partition-coefficient data on that justification, this is no longer sufficient as new methods to assess POW have become available.

Standard tests for determining partition coefficient

OECD 107 is the standard method for determining POW but is only applicable for substances with log POW between -2 to 4, as the shake-flask methodology can force octanol microdroplets into the aqueous phase leading to the overestimation of substance in water for test substances with a log POW >4.

POW assessment of gases can also be challenging but, by using variations of the standard OECD 107 test method, this can be successfully achieved. 

Rationale for using the OECD 123 to determine POW

OECD 123 reduces the POW artefacts caused by microdroplets – the main drawback to OECD 107. It does so by using a slow-stirring method rather than the shake-flask method used in OECD 107.

Utilizing the OECD 123 test in practice

For OECD 123, the test substance is equilibrated with water and octanol in a stirred chamber. Once equilibrium between the phases is reached, POW is measured directly in the same way as for OECD 107. The time required to reach equilibrium varies depending on the substance’s hydrophobicity; the more hydrophobic the substance, the longer it takes to reach equilibrium.

Implications for crop or chemical active substance registrations or reapprovals

Historically, if a substance had a POW >4, then no further quantification was made and the data waived. However, by using the stirred method, POW can now be estimated for these substances and data waivers may now be perceived as data gaps. This endpoint may need to be reassessed with this test to align with current standards.

Flammability and autoignition: selecting the right testing approach to satisfy the regulatory needs

The physical chemical endpoints of flammability and autoignition are important for safety assessment and for classification and labeling of active substances. For many years, the EU Commission methods (EU A9–A17) were the internationally recognized standard tests used for assessment of flammability endpoints and they are still valid and in use today. However, most of those EU tests have been superseded by test methods defined in the Manual of Tests and Criteria of the United Nations Recommendations on the Transport of Dangerous Goods, Model Regulations and the Globally Harmonized System of Classification and Labelling of Chemicals (GHS). Two key endpoints of note are flammability and autoignition of a solid, both of which influence the classification of a solid substance as flammable. The section below looks at the differences in the EU and UN methods used to assess those endpoints.

Test options for determining flammability of a solid: EU method A10 and UN method N.1

Both the EU A10 and UN N.1 methods are similar and involve the construction of a 25-cm long mold of the solid test substance, which is then set on fire at one end. The time taken to burn from end to end is measured and used to classify flammability. For UN N.1 test, there is a wet zone approximately halfway along the length of the mold to test if the flame can jump the wetted area.

Test options for determining autoignition of a solid: EU method A16 and UN method N.4

Again, the EU A16 and UN N.4 methods are designed to assess the self-combustion potential of the substance and follow a generally similar design. The test substance is packed into an open-topped mesh cube and suspended in an oven where it is heated. Under EU method A16, the oven temperature is increased to 400°C and, if the substance ignites, a sharp increase in temperature of the substance above that of the oven is observed – the self-ignition temperature. Under UN method N.4, self-ignition is confirmed by heating the cube at 140°C for 24 hours. A positive result is indicated by self-ignition or if the temperature of the substance exceeds that of the oven by 60°C.

Implications for crop or chemical active substance registrations or reapprovals

Although key flammability endpoints assessed by these different methods are the same, the tests used to assess them are subtly, but importantly, different. It is essential to appreciate the regulatory context for assessing physical chemical endpoints, so that the most appropriate test methods can be used.

Conclusions

Physical chemistry is fundamental to the development and subsequent approval of a crop or chemical active substance. Determining the best way to assess key physical chemistry parameters requires an understanding of both the nature of the test material and of the regulatory context in which it will be used. It is tempting to categorize physical chemistry testing as the simple, boring stuff you just need to work through, but it is vitally important. For many older substances, these basic properties may have been determined in non-GLP facilities using tests that may no longer match the latest test guidance. For both new and old substances, fresh thinking on physical chemistry endpoints and tests is becoming more important, especially as the regulatory landscape changes. A good example is the EU regulation on nanomaterials, where the physical chemistry parameters of particle size, shape and surface chemistry all play an important part in nanoform classification. So, cherish the boring and give physical chemistry more attention – it is the foundation on which your substance’s success is built.

For similar material on this subject, read our case study, Physicochemical Testing Case Study – Handling Difficult Substances – Gases or visit our website.