EN Official Journal of the European Union L 220/1

COMMISSION REGULATION (EC) No 761/2009

of 23 July 2009

amending, for the purpose of its adaptation to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)

(Text with EEA relevance)

THE COMMISSION OF THE EUROPEAN COMMUNITIES,

Having regard to the Treaty establishing the European Community,

Having regard to Regulation (EC) No 1907/2006 of 18 December 2006 of the European Parliament and of the Council concerning the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), establishing a European Chemicals Agency, amending Directive 1999/45/EC and repealing Council Regulation (EEC) No 793/93 and Commission Regulation (EC) No 1488/94 as well as Council Directive 76/769/EEC and Commission Directives 91/155/EEC, 93/67/EEC, 93/105/EC and 2000/21/EC (1), and in particular Article 13(3) thereof,

Whereas:

(1) Commission Regulation (EC) No 440/2008 (2) contains the test methods for the purposes of the determination of the physico-chemical properties, toxicity and eco-toxicity of substances to be applied for the purposes of Regulation (EC) No 1907/2006.

(2) It is necessary to update Regulation (EC) No 440/2008 to include changes to certain test methods and to include several new test methods adopted by the OECD. Stakeholders have been consulted on this proposal. Those amendments adapt the methods in question to scientific and technical progress.

(3) The provisions concerning vapour pressure to include the new effusion method should be revised.

(4) It is necessary to add a new method to measure length weighted geometric mean diameter of fibres.

(5) It is appropriate to update Regulation (EC) No 440/2008 to include with priority a new in vitro test method for skin irritation, in order to obtain a reduction of the number of animals to be used for experimental purposes, in accordance with Council Directive 86/609/EEC of 24 November 1986 on the approximation of laws, regulations and administrative provisions of the Member States regarding the protection of animals used for experimental and other scientific purposes (3). Although a draft in vitro test method for skin irritation is still under discussion within the OECD, it is appropriate in this exceptional case to include method B 46 in this Regulation. Method B 46 should be updated as soon as possible once an agreement has been reached within the OECD or if further information justifying such a revision becomes available.

(6) The provisions concerning the algal inhibition test need to be revised to include additional species and to meet the requirements for hazard assessment and classification of chemicals.

(7) It is necessary to add a new method to measure aerobic mineralisation in surface water, by virtue of a simulation biodegradation test and a new method to assess toxicity to the genus Lemna, by virtue of a growth inhibition test.

(8) Regulation (EC) No 440/2008 should therefore be amended accordingly.

(9) The measures provided for in this Regulation are in accordance with the opinion of the Committee established under Article 133 of Regulation (EC) No 1907/2006,

HAS ADOPTED THIS REGULATION:

Article 1

The Annex to Regulation (EC) No 440/2008 is amended as follows:

1. Part A is amended as follows: (a) Chapter A.4 is replaced by Chapter A.4 as set out in Annex I to this Regulation; (b) Chapter A.22 as set out in Annex II to this Regulation is added;

2. Part B is amended as follows: Chapter B.46 as set out in Annex III to this Regulation is added;

3. Part C is amended as follows: (a) Chapter C.3 is replaced by Chapter C.3 as set out in Annex IV to this Regulation; (b) Chapters C.25 and C.26 as set out in Annexes V and VI to this Regulation are added.

Article 2

This Regulation shall enter into force on the third day following its publication in the Official Journal of the European Union.

This Regulation shall be binding in its entirety and directly applicable in all Member States. Done at Brussels, 23 July 2009. For the Commission Stavros DIMAS Member of the Commission

(1) OJ L 396, 30.12.2006, p. 1.

(2) OJ L 142, 31.5.2008, p. 1.

(3) OJ L 358, 18.12.1986, p. 1.

ANNEX I A.4. VAPOUR PRESSURE 1. METHOD This method is equivalent to OECD TG 104 (2004). 1.1. INTRODUCTION This revised version of method A.4(1) includes one additional method; Effusion method: isothermal thermogravimetry, designed for substances with very low pressures (down to 10–10 Pa). In the light of needs for procedures, especially in relation to obtaining vapour pressure for substances with low vapour pressure, other procedures of this method are re-evaluated with respect to other applicability ranges. At the thermodynamic equilibrium the vapour pressure of a pure substance is a function of temperature only. The fundamental principles are described elsewhere (2)(3). No single measurement procedure is applicable to the entire range of vapour pressures from less than 10–10 to 105 Pa. Eight methods for measuring vapour pressure are included in this method which can be applied in different vapour pressure ranges. The various methods are compared as to application and measuring range in Table 1. The methods can only be applied for compounds that do not decompose under the conditions of the test. In cases where the experimental methods cannot be applied due to technical reasons, the vapour pressure can also be estimated, and a recommended estimation method is set out in the Appendix. 1.2. DEFINITIONS AND UNITS The vapour pressure of a substance is defined as the saturation pressure above a solid or liquid substance. The SI unit of pressure, which is the pascal (Pa), should be used. Other units which have been employed historically are given hereafter, together with their conversion factors: 1 Torr = 1 mm Hg = 1,333 × 102 Pa 1 atmosphere = 1,013 × 105 Pa 1 bar = 105 Pa The SI unit of temperature is the kelvin (K). The conversion of degrees Celsius to kelvin is according to the formula: T = t + 273,15 where, T is the kelvin or thermodynamic temperature and t is the Celsius temperature. Table 1 Measuring method Substances Estimated repeatability Estimated reproducibility Recommended range Solid Liquid Dynamic method Low melting Yes up to 25 % 1 to 5 % up to 25 % 1 to 5 % 103 Pa to 2 × 103 Pa 2 × 103 Pa to 105 Pa Static method Yes Yes 5 to 10 % 5 to 10 % 10 Pa to 105 Pa 10–2 Pa to 105 Pa (1) Isoteniscope method Yes Yes 5 to 10 % 5 to 10 % 102 Pa to 105 Pa Effusion method: vapour pressure balance Yes Yes 5 to 20 % up to 50 % 10–3 to 1 Pa Effusion method: Knudsen cell Yes Yes 10 to 30 % — 10–10 to 1 P Effusion method: isothermal thermogravimetry Yes Yes 5 to 30 % up to 50 % 10–10 to 1 Pa Gas saturation method Yes Yes 10 to 30 % up to 50 % 10–10 to 103 Pa Spinning rotor method Yes Yes 10 to 20 % — 10–4 to 0,5 Pa 1.3. PRINCIPLE OF THE TEST In general, the vapour pressure is determined at various temperatures. In a limited temperature range, the logarithm of the vapour pressure of a pure substance is a linear function of the inverse of the thermodynamic temperature according to the simplified Clapeyron-Clausius equation: where: p = the vapour pressure in pascals ΔHv = the heat of vaporisation in J mol–1 R = the universal gas constant, 8,314 J mol–1 K–1 T = the temperature in K 1.4. REFERENCE SUBSTANCES Reference substances do not need to be employed. They serve primarily to check the performance of a method from time to time as well as to allow comparison between results of different methods. 1.5. DESCRIPTION OF THE METHOD 1.5.1. Dynamic method (Cottrell’s method) 1.5.1.1. Principle The vapour pressure is determined by measuring the boiling temperature of the substance at various specified pressures between roughly 103 and 105 Pa. This method is also recommended for the determination of the boiling temperature. For that purpose it is useful up to 600 K. The boiling temperatures of liquids are approximately 0,1 °C higher at a depth of 3 to 4 cm than at the surface because of the hydrostatic pressure of the column of liquid. In Cottrell’s method (4) the thermometer is placed in the vapour above the surface of the liquid and the boiling liquid is made to pump itself continuously over the bulb of the thermometer. A thin layer of liquid which is in equilibrium with vapour at atmospheric pressure covers the bulb. The thermometer thus reads the true boiling point, without errors due to superheating or hydrostatic pressure. The pump originally employed by Cottrell is shown in figure 1. Tube A contains the boiling liquid. A platinum wire B sealed into the bottom facilitates uniform boiling. The side tube C leads to a condenser, and the sheath D prevents the cold condensate from reaching the thermometer E. When the liquid in A is boiling, bubbles and liquid trapped by the funnel are poured via the two arms of the pump F over the bulb of the thermometer. Figure 1 Figure 2 Cottrell pump (4) A: Thermocouple B: Vacuum buffer volume C: Pressure gauge D: Vacuum E: Measuring point F: Heating element c.a. 150 W 1.5.1.2. Apparatus A very accurate apparatus, employing the Cottrell principle, is shown in figure 2. It consists of a tube with a boiling section in the lower part, a cooler in the middle part, and an outlet and flange in the upper part. The Cottrell pump is placed in the boiling section which is heated by means of an electrical cartridge. The temperature is measured by a jacketed thermocouple, or resistance thermometer inserting through the flange at the top. The outlet is connected to the pressure regulation system. The latter consists of a vacuum pump, a buffer volume, a manostat for admitting nitrogen for pressure regulation and manometer. 1.5.1.3. Procedure The substance is placed in the boiling section. Problems may be encountered with non-powder solids but these can sometimes be solved by heating the cooling jacket. The apparatus is sealed at the flange and the substance degassed. Frothing substances cannot be measured using this method. The lowest desired pressure is then set and the heating is switched on. At the same time, the temperature sensor is connected to a recorder. Equilibrium is reached when a constant boiling temperature is recorded at constant pressure. Particular care must be taken to avoid bumping during boiling. In addition, complete condensation must occur on the cooler. When determining the vapour pressure of low melting solids, care should be taken to prevent the condenser from blocking. After recording this equilibrium point, a higher pressure is set. The process is continued in this manner until 105 Pa has been reached (approximately 5 to 10 measuring points in all). As a check, equilibrium points must be repeated at decreasing pressures. 1.5.2. Static method 1.5.2.1. Principle In the static method (5), the vapour pressure at thermodynamic equilibrium is determined at a specified temperature. This method is suitable for substances and multicomponent liquids and solids in the range from 10–1 to 105 Pa and, provided care is taken, also in the range 1 to 10 Pa. 1.5.2.2. Apparatus The equipment consists of a constant-temperature bath (precision of ±0,2 K), a container for the sample connected to a vacuum line, a manometer and a system to regulate the pressure. The sample chamber (figure 3a) is connected to the vacuum line via a valve and a differential manometer (U-tube containing a suitable manometer fluid) which serves as zero indicator. Mercury, silicones and phthalates are suitable for use in the differential manometer, depending on the pressure range and the chemical behaviour of the test substance. However, based on environmental concerns, the use of mercury should be avoided, if possible. The test substance must not dissolve noticeably in, or react with, the U-tube fluid. A pressure gauge can be used instead of a U-tube (figure 3b). For the manometer, mercury can be used in the range from normal pressure down to 102 Pa, while silicone fluids and phthalates are suitable for use below 102 Pa down to 10 Pa. There are other pressure gauges which can be used below 102 Pa and heatable membrane capacity manometers can even be used at below 10–1 Pa. The temperature is measured on the outside wall of the vessel containing the sample or in the vessel itself. 1.5.2.3. Procedure Using the apparatus as described in figure 3a, fill the U-tube with the chosen liquid, which must be degassed at an elevated temperature before readings are taken. The test substance is placed in the apparatus and degassed at reduced temperature. In the case of a multiple-component sample, the temperature should be low enough to ensure that the composition of the material is not altered. Equilibrium can be established more quickly by stirring. The sample can be cooled with liquid nitrogen or dry ice, but care should be taken to avoid condensation of air or pump-fluid. With the valve over the sample vessel open, suction is applied for several minutes to remove the air. If necessary, the degassing operation is repeated several times. Figure 3a Figure 3b When the sample is heated with the valve closed, the vapour pressure increases. This alters the equilibrium of the fluid in the U-tube. To compensate for this, nitrogen or air is admitted to the apparatus until the differential pressure indicator is at zero again. The pressure required for this can be read off the manometer or off an instrument of higher precision. This pressure corresponds to the vapour pressure of the substance at the temperature of the measurement. Using the apparatus described in figure 3b, the vapour pressure is read off directly. The vapour pressure is determined at suitably small temperature intervals (approximately 5 to 10 measuring points in all) up to the desired temperature maximum. Low-temperature readings must be repeated as a check. If the values obtained from the repeated readings do not coincide with the curve obtained for increasing temperature, this may be due to one of the following situations: (i) the sample still contains air (e.g. in the case of highly viscous materials) or low-boiling substances which is or are released during heating; (ii) the substance undergoes a chemical reaction in the temperature range investigated (e.g. decomposition, polymerisation). 1.5.3. Isoteniscope Method 1.5.3.1. Principle The isoteniscope (6) is based on the principle of the static method. The method involves placing a sample in a bulb maintained at constant temperature and connected to a manometer and a vacuum pump. Impurities more volatile than the substance are removed by degassing at reduced pressure. The vapour pressure of the sample at selected temperatures is balanced by a known pressure of inert gas. The isoteniscope was developed to measure the vapour pressure of certain liquid hydrocarbons but it is appropriate for the investigation of solids as well. The method is usually not suitable for multicomponent systems. Results are subject to only slight errors for samples containing non-volatile impurities. The recommended range is 102 to 105 Pa. 1.5.3.2. Apparatus An example of a measuring device is shown in figure 4. A complete description can be found in ASTM D 2879-86 (6). 1.5.3.3. Procedure In the case of liquids, the substance itself serves as the fluid in the differential manometer. A quantity of the liquid, sufficient to fill the bulb and the short leg of the manometer, is put in the isoteniscope. The isoteniscope is attached to a vacuum system and evacuated, then filled by nitrogen. The evacuation and purge of the system is repeated twice to remove residual oxygen. The filled isoteniscope is placed in a horizontal position so that the sample spreads out into a thin layer in the sample bulb and manometer. The pressure of the system is reduced to 133 Pa and the sample is gently warmed until it just boils (removal of dissolved gases). The isoteniscope is then placed so that the sample returns to the bulb and fills the short leg of the manometer. The pressure is maintained at 133 Pa. The drawn-out tip of the sample bulb is heated with a small flame until the sample vapour released expands sufficiently to displace part of the sample from the upper part of the bulb and manometer arm into the manometer, creating a vapour-filled, nitrogen-free space. The isoteniscope is then placed in a constant temperature bath, and the pressure of the nitrogen is adjusted until it equals that of the sample. At the equilibrium, the pressure of the nitrogen equals the vapour pressure of the substance. Figure 4 In the case of solids, and depending on the pressure and temperature ranges, manometer liquids such as silicon fluids or phthalates are used. The degassed manometer liquid is put in a bulge provided on the long arm of the isoteniscope. Then the solid to be investigated is placed in the sample bulb and is degassed at an elevated temperature. After that, the isoteniscope is inclined so that the manometer liquid can flow into the U-tube. 1.5.4. Effusion method: vapour pressure balance (7) 1.5.4.1. Principle A sample of the test substance is heated in a small furnace and placed in an evacuated bell jar. The furnace is covered by a lid which carries small holes of known diameters. The vapour of the substance, escaping through one of the holes, is directed onto a balance pan of a highly sensitive balance which is also enclosed in the evacuated bell jar. In some designs the balance pan is surrounded by a refrigeration box, providing heat dissipation to the outside by thermal conduction, and is cooled by radiation so that the escaping vapour condenses on it. The momentum of the vapour jet acts as a force on the balance. The vapour pressure can be derived in two ways: directly from the force on the balance pan and also from the evaporation rate using the Hertz-Knudsen equation (2): where: G = evaporation rate (kg s–1 m–2) M = molar mass (g mol–1) T = temperature (K) R = universal gas constant (J mol–1 K–1) P = vapour pressure (Pa) The recommended range is 10–3 to 1 Pa. 1.5.4.2. Apparatus The general principle of the apparatus is illustrated in figure 5. Figure 5 A: Base plate F: Refrigeration box and cooling bar B: Moving coil instrument G: Evaporator furnace C: Bell jar H: Dewar flask with liquid nitrogen D: Balance with scale pan I: Measurement of temperature of sample E: Vacuum measuring device J: Test Substance 1.5.5. Effusion method: Knudsen cell 1.5.5.1. Principle The method is based on the estimation of the mass of test substance flowing out per unit of time of a Knudsen cell (8) in the form of vapour, through a micro-orifice under ultra-vacuum conditions. The mass of effused vapour can be obtained either by determining the loss of mass of the cell or by condensing the vapour at low temperature and determining the amount of volatilised substance using chromatography. The vapour pressure is calculated by applying the Hertz-Knudsen relation (see section 1.5.4.1) with correction factors that depend on parameters of the apparatus (9). The recommended range is 10–10 to 1 Pa (10)(11)(12)(13)(14). 1.5.5.2. Apparatus The general principle of the apparatus is illustrated in figure 6. Figure 6 1: Connection to vacuum 7: Threaded lid 2: Wells from platinum resistance thermometer or temperature measurement and control 8: Butterfly nuts 3: Lid for vacuum tank 9: Bolts 4: O-ring 10: Stainless steel effusion cells 5: Aluminum vacuum tank 11: Heater cartridge 6: Device for installing and removing the effusion cells 1.5.6. Effusion method: isothermal thermogravimetry 1.5.6.1. Principle The method is based on the determination of accelerated evaporation rates for the test substance at elevated temperatures and ambient pressure using thermogravimetry (10)(15)(16)(17)(18)(19)(20). The evaporation rates vT result from exposing the selected compound to a slowly flowing inert gas atmosphere, and monitoring the weight loss at defined isothermal temperatures T in Kelvin over appropriate periods of time. The vapour pressures pT are calculated from the vT values by using the linear relationship between the logarithm of the vapour pressure and the logarithm of the evaporation rate. If necessary, an extrapolation to temperatures of 20 and 25 °C can be made by regression analysis of log pT vs. 1/T. This method is suitable for substances with vapour pressures as low as 10–10 Pa (10–12 mbar) and with purity as close as possible to 100 % to avoid the misinterpretation of measured weight losses. 1.5.6.2. Apparatus The general principle of the experimental set-up is shown in figure 7. Figure 7 The sample carrier plate, hanging on a microbalance in a temperature controlled chamber, is swept by a stream of dry nitrogen gas which carries the vaporised molecules of the test substance away. After leaving the chamber, the gas stream is purified by a sorption unit. 1.5.6.3. Procedure The test substance is applied to the surface of a roughened glass plate as a homogeneous layer. In the case of solids, the plate is wetted uniformly by a solution of the substance in a suitable solvent and dried in an inert atmosphere. For the measurement, the coated plate is hung into the thermogravimetric analyser and subsequently its weight loss is measured continuously as a function of time. The evaporation rate vT at a definite temperature is calculated from the weight loss Δm of the sample plate by where F is the surface area of the coated test substances, normally the surface area of the sample plate, and t is the time for weight loss Δm. The vapour pressure pT is calculated on the basis of its function of evaporation rate vT: Log pT = C + D · log vT where C and D are constants specific for the experimental arrangement used, depending on the diameter of the measurement chamber and on the gas flow rate. These constants must be determined once by measuring a set of compounds with known vapour pressure and regressing log pT vs. log vT (11)(21)(22). The relationship between the vapour pressure pT and the temperature T in Kelvin is given by Log pT = A + B · 1/T where A and B are constants obtained by regressing log pT vs. 1/T. With this equation, the vapour pressure can be calculated for any other temperature by extrapolation. 1.5.7. Gas saturation method (23) 1.5.7.1. Principle Inert gas is passed, at room temperature and at a known flow rate, through or over a sample of the test substance, slowly enough to ensure saturation. Achieving saturation in the gas phase is of critical importance. The transported substance is trapped, generally using a sorbent, and its amount is determined. As an alternative to vapour trapping and subsequent analysis, in-train analytical techniques, like gas chromatography, may be used to determine quantitatively the amount of material transported. The vapour pressure is calculated on the assumption that the ideal gas law is obeyed and that the total pressure of a mixture of gases is equal to the sum of the pressures of the component gases. The partial pressure of the test substance, i.e. the vapour pressure, is calculated from the known total gas volume and from the weight of the material transported. The gas saturation procedure is applicable to solid or liquid substances. It can be used for vapour pressures down to 10–10 Pa (10)(11)(12)(13)(14). The method is most reliable for vapour pressures below 103 Pa. Above 103 Pa, the vapour pressures are generally overestimated, probably due to aerosol formation. Since the vapour pressure measurements are made at room temperature, the need to extrapolate data from high temperatures is not necessary and high temperature extrapolation, which can often cause serious errors, is avoided. 1.5.7.2. Apparatus The procedure requires the use of a constant-temperature box. The sketch in figure 8 shows a box containing three solid and three liquid sample holders, which allow for the triplicate analysis of either a solid or a liquid sample. The temperature is controlled to ±0,5 °C or better. Figure 8 In general, nitrogen is used as an inert carrier gas but, occasionally, another gas may be required (24). The carrier gas must be dry. The gas stream is split into 6 streams, controlled by needle valves (approximately 0,79 mm orifice), and flows into the box via 3,8 mm i.d. copper tubing. After temperature equilibration, the gas flows through the sample and the sorbent trap and exists from the box. Solid samples are loaded into 5 mm i.d. glass tubing between glass wool plugs (see Figure 9). Figure 10 shows a liquid sample holder and sorbent system. The most reproducible method for measuring the vapour pressure of liquids is to coat the liquid on glass beads or on an inert sorbent such as silica, and to pack the holder with these beads. As an alternative, the carrier gas may be made to pass a coarse frit and bubble through a column of the liquid test substance. Figure 9 Figure 10 The sorbent system contains a front and a backup sorbent section. At very low vapour pressures, only small amounts are retained by the sorbent and the adsorption on the glass wool and the glass tubing between the sample and the sorbent may be a serious problem. Traps cooled with solid CO2 are another efficient way for collecting the vaporised material. They do not cause any back pressure on the saturator column and it is also easy to quantitatively remove the trapped material. 1.5.7.3. Procedure The flow rate of the effluent carrier gas is measured at room temperature. The flow rate is checked frequently during the experiment to assure that there is an accurate value for the total volume of carrier gas. Continuous monitoring with a mass flow-meter is preferred. Saturation of the gas phase may require considerable contact time and hence quite low gas flow rates (25). At the end of the experiment, both the front and backup sorbent sections are analysed separately. The compound on each section is desorbed by adding a solvent. The resulting solutions are analysed quantitatively to determine the weight desorbed from each section. The choice of the analytical method (also the choice of sorbent and desorbing solvent) is dictated by the nature of the test material. The desorption efficiency is determined by injecting a known amount of sample onto the sorbent, desorbing it and analysing the amount recovered. It is important to check the desorption efficiency at or near the concentration of the sample under the test conditions. To assure that the carrier gas is saturated with the test substance, three different gas flow rates are used. If the calculated vapour pressure shows no dependence on flow rate, the gas is assumed to be saturated. The vapour pressure is calculated through the equation: where: p = vapour pressure (Pa) W = mass of evaporated test substance (g) V = volume of saturated gas (m3) R = universal gas constant 8,314 (J mol–1 K–1) T = temperature (K) M = molar mass of test substance (g mol–1) Measured volumes must be corrected for pressure and temperature differences between the flow meter and the saturator. 1.5.8. Spinning rotor 1.5.8.1. Principle This method uses a spinning rotor viscosity gauge, in which the measuring element is a small steel ball which, suspended in a magnetic field, is made to spin by rotating fields (26)(27)(28). Pick-up coils allow its spinning rate to be measured. When the ball has reached a given rotational speed, usually about 400 revolutions per second, energising is stopped and deceleration, due to gas friction, takes place. The drop of rotational speed is measured as a function of time. The vapour pressure is deduced from the pressure-dependent slow-down of the steel ball. The recommended range is 10–4 to 0,5 Pa. 1.5.8.2. Apparatus A schematic drawing of the experimental set-up is shown in figure 11. The measuring head is placed in a constant-temperature enclosure, regulated within 0,1 °C. The sample container is placed in a separate enclosure, also regulated within 0,1 °C. All other parts of the set-up are kept at a higher temperature to prevent condensation. The whole apparatus is connected to a high-vacuum system. Figure 11 2. DATA AND REPORTING 2.1. DATA The vapour pressure from any of the preceding methods should be determined for at least two temperatures. Three or more are preferred in the range from 0 to 50 °C, in order to check the linearity of the vapour pressure curve. In case of Effusion method (Knudsen cell and isothermal thermogravimetry) and Gas saturation method, 120 to 150 °C is recommended for the measuring temperature range instead of 0 to 50 °C. 2.2. TEST REPORT The test report must include the following information: — method used, — precise specification of the substance (identity and impurities) and preliminary purification step, if any, — at least two vapour pressure and temperature values — and preferably three or more — required in the range from 0 to 50 °C (or 120 to 150 °C), — at least one of the temperatures should be at or below 25 °C, if technically possible according to the chosen method, — all original data, — a log p versus 1/T curve, — an estimate of the vapour pressure at 20 or 25 °C. If a transition (change of state, decomposition) is observed, the following information should be noted: — nature of the change, — temperature at which the change occurs at atmospheric pressure, — vapour pressure at 10 and 20 °C below the transition temperature and 10 and 20 °C above this temperature (unless the transition is from solid to gas). All information and remarks relevant for the interpretation of results have to be reported, especially with regard to impurities and physical state of the substance. 3. LITERATURE (1) Official Journal of the European Communities L 383 A, 26-47 (1992). (2) Ambrose, D. (1975). Experimental Thermodynamics, Vol. II, Le Neindre, B., and Vodar, B., Eds., Butterworths, London. (3) Weissberger R., ed. (1959). Technique of Organic Chemistry, Physical Methods of Organic Chemistry, 3rd ed., Vol. I, Part I. Chapter IX, Interscience Publ., New York. (4) Glasstone, S. (1946). Textbook of Physical Chemistry, 2nd ed., Van Nostrand Company, New York. (5) NF T 20-048 AFNOR (September 1985). Chemical products for industrial use — Determination of vapour pressure of solids and liquids within a range from 10–1 to 105 Pa — Static method. (6) ASTM D 2879-86, Standard test method for vapour pressure — temperature relationship and initial decomposition temperature of liquids by isoteniscope. (7) NF T 20-047 AFNOR (September 1985). Chemical products for industrial use — Determination of vapour pressure of solids and liquids within range from 10–3 to 1 Pa — Vapour pressure balance method. (8) Knudsen, M. (1909). Ann. Phys. Lpz., 29, 1979; (1911), 34, 593. (9) Ambrose, D., Lawrenson, I.J., Sprake, C.H.S. (1975). J. Chem. Thermodynamics 7, 1173. (10) Schmuckler, M.E., Barefoot, A.C., Kleier, D.A., Cobranchi, D.P. (2000), Vapor pressures of sulfonylurea herbicides; Pest Management Science 56, 521-532. (11) Tomlin, C.D.S. (ed.), The Pesticide Manual, Twelfth Edition (2000). (12) Friedrich, K., Stammbach, K., Gas chromatographic determination of small vapour pressures determination of the vapour pressures of some triazine herbicides. J. Chromatog. 16 (1964), 22-28. (13) Grayson, B.T., Fosbraey, L.A., Pesticide Science 16 (1982), 269-278. (14) Rordorf, B.F., Prediction of vapor pressures, boiling points and enthalpies of fusion for twenty-nine halogenated dibenzo-p-dioxins, Thermochimia Acta 112 Issue 1 (1987), 117-122. (15) Gückel, W., Synnatschke, G., Ritttig, R., A Method for Determining the Volatility of Active Ingredients Used in Plant Protection; Pesticide Science 4 (1973) 137-147. (16) Gückel, W., Synnatschke, G., Ritttig, R., A Method for Determining the Volatility of Active Ingredients Used in Plant Protection II. Application to Formulated Products; Pesticide Science 5 (1974) 393-400. (17) Gückel, W., Kaestel, R., Lewerenz, J., Synnatschke, G., A Method for Determining the Volatility of Active Ingredients Used in Plant Protection. Part III: The Temperature Relationship between Vapour Pressure and Evaporation Rate; Pesticide Science 13 (1982) 161-168. (18) Gückel, W., Kaestel, R., Kroehl, T., Parg, A., Methods for Determining the Vapour Pressure of Active Ingredients Used in Crop Protection. Part IV: An Improved Thermogravimetric Determination Based on Evaporation Rate; Pesticide Science 45 (1995) 27-31. (19) Kroehl, T., Kaestel, R., Koenig, W., Ziegler, H., Koehle, H., Parg, A., Methods for Determining the Vapour Pressure of Active Ingredients Used in Crop Protection. Part V: Thermogravimetry Combined with Solid Phase MicroExtraction (SPME); Pesticide Science, 53 (1998) 300-310. (20) Tesconi, M., Yalkowsky, S.H., A Novel Thermogravimetric Method for Estimating the Saturated Vapor Pressure of Low-Volatility Compounds; Journal of Pharmaceutical Science 87(12) (1998) 1512-20. (21) Lide, D.R. (ed.), CRC Handbook of Chemistry and Physics, 81st ed. (2000), Vapour Pressure in the Range — 25 °C to 150 °C. (22) Meister, R.T. (ed.), Farm Chemicals Handbook, Vol. 88 (2002). (23) 40 CFR, 796. (1993). pp 148-153, Office of the Federal Register, Washington DC. (24) Rordorf B.F. (1985). Thermochimica Acta 85, 435. (25) Westcott et al. (1981). Environ. Sci. Technol. 15, 1375. (26) Messer G., Röhl, P., Grosse G., and Jitschin W. (1987). J. Vac. Sci. Technol. (A), 5(4), 2440. (27) Comsa G., Fremerey J.K., and Lindenau, B. (1980). J. Vac. Sci. Technol. 17(2), 642. (28) Fremerey, J.K. (1985). J. Vac. Sci. Technol. (A), 3(3), 1715. (1) When using a capacitance manometer Appendix Estimation method INTRODUCTION Estimated values of the vapour pressure can be used: — for deciding which of the experimental methods is appropriate, — for providing an estimate or limit value in cases where the experimental method cannot be applied due to technical reasons. ESTIMATION METHOD The vapour pressure of liquids and solids can be estimated by use of the modified Watson correlation (a). The only experimental data required is the normal boiling point. The method is applicable over the pressure range from 105 Pa to 10–5 Pa. Detailed information on the method is given in ‘Handbook of Chemical Property Estimation Methods’ (b). See also OECD Environmental Monograph No.67 (c). CALCULATION PROCEDURE The vapour pressure is calculated as follows: where: T = temperature of interest Tb = normal boiling point Pvp = vapour pressure at temperature T ΔHvb = heat of vaporisation ΔZb = compressibility factor (estimated at 0,97) m = empirical factor depending on the physical state at the temperature of interest Further, where, KF is an empirical factor considering the polarity of the substance. For several compound types, KF factors are listed in reference (b). Quite often, data are available in which a boiling point at reduced pressure is given. In such a case, the vapour pressure is calculated as follows: where, T1 is the boiling point at the reduced pressure P1. REPORT When using the estimation method, the report shall include a comprehensive documentation of the calculation. LITERATURE (a) Watson, K.M. (1943). Ind. Eng. Chem, 35, 398. (b) Lyman, W.J., Reehl, W.F., Rosenblatt, D.H. (1982). Handbook of Chemical Property Estimation Methods, McGraw-Hill. (c) OECD Environmental Monograph No.67. Application of Structure-Activity Relationships to the Estimation of Properties Important in Exposure Assessment (1993).

ANNEX II A.22. LENGTH WEIGHTED GEOMETRIC MEAN DIAMETER OF FIBRES 1. METHOD 1.1. INTRODUCTION This method describes a procedure to measure the Length Weighted Geometric Mean Diameter (LWGMD) of bulk Man Made Mineral Fibres (MMMF). As the LWGMD of the population will have a 95 % probability of being between the 95 % confidence levels (LWGMD ± two standard errors) of the sample, the value reported (the test value) will be the lower 95 % confidence limit of the sample (i.e. LWGMD — 2 standard errors). The method is based on an update (June 1994) of a draft HSE industry procedure agreed at a meeting between ECFIA and HSE at Chester on 26/9/93 and developed for and from a second inter-laboratory trial (1, 2). This measurement method can be used to characterise the fibre diameter of bulk substances or products containing MMMFs including refractory ceramic fibres (RCF), man-made vitreous fibres (MMVF), crystalline and polycrystalline fibres. Length weighting is a means of compensating for the effect on the diameter distribution caused by the breakage of long fibres when sampling or handling the material. Geometric statistics (geometric mean) are used to measure the size distribution of MMMF diameters because these diameters usually have size distributions that approximate to log normal. Measuring length as well as diameter is both tedious and time consuming but, if only those fibres that touch an infinitely thin line on a SEM field of view are measured, then the probability of selecting a given fibre is proportional to its length. As this takes care of the length in the length weighting calculations, the only measurement required is the diameter and the LWGMD-2SE can be calculated as described. 1.2. DEFINITIONS Particle: An object with a length to width ratio of less than 3:1. Fibre: An object with a length to with ratio (aspect ratio) of at least 3:1. 1.3. SCOPE AND LIMITATIONS The method is designed to look at diameter distributions which have median diameters from 0,5 μm to 6 μm. Larger diameters can be measured by using lower SEM magnifications but the method will be increasingly limited for finer fibre distributions and a TEM (transmission electron microscope) measurement is recommended if the median diameter is below 0,5 μm. 1.4. PRINCIPLE OF THE TEST METHOD A number of representative core samples are taken from the fibre blanket or from loose bulk fibre. The bulk fibres are reduced in length using a crushing procedure and a representative sub-sample dispersed in water. Aliquots are extracted and filtered through a 0,2 μm pore size, polycarbonate filter and prepared for examination using scanning electron microscope (SEM) techniques. The fibre diameters are measured at a screen magnification of × 10 000 or greater (1) using a line intercept method to give an unbiased estimate of the median diameter. The lower 95 % confidence interval (based on a one sided test) is calculated to give an estimate of the lowest value of the geometric mean fibre diameter of the material. 1.5. DESCRIPTION OF THE TEST METHOD 1.5.1. Safety/precautions Personal exposure to airborne fibres should be minimised and a fume cupboard or glove box should be used for handling the dry fibres. Periodic personal exposure monitoring should be carried out to determine the effectiveness of the control methods. When handling MMMF’s disposable gloves should be worn to reduce skin irritation and to prevent cross-contamination. 1.5.2. Apparatus/equipment — Press and dyes (capable of producing 10 MPa). — 0,2 μm pore size polycarbonate capillary pore filters (25 mm diameter). — 5 μm pore size cellulose ester membrane filter for use as a backing filter. — Glass filtration apparatus (or disposable filtration systems) to take 25 mm diameter filters (e.g. Millipore glass microanalysis kit, type No XX10 025 00). — Freshly distilled water that has been filtered through a 0,2 μm pore size filter to remove micro-organisms. — Sputter coater with a gold or gold/palladium target. — Scanning electron microscope capable of resolving down to 10 nm and operating at × 10 000 magnification. — Miscellaneous: spatulas, type 24 scalpel blade, tweezers, SEM tubes, carbon glue or carbon adhesive tape, silver dag. — Ultrasonic probe or bench top ultrasonic bath. — Core sampler or cork borer, for taking core samples from MMMF blanket. 1.5.3. Test Procedure 1.5.3.1. Sampling For blankets and bats a 25 mm core sampler or cork borer is used to take samples of the cross-section. These should be equally spaced across the width of a small length of the blanket or taken from random areas if long lengths of the blanket are available. The same equipment can be used to extract random samples from loose fibre. Six samples should be taken when possible, to reflect spatial variations in the bulk material. The six core samples should be crushed in a 50 mm diameter dye at 10 MPa. The material is mixed with spatula and re-pressed at 10 MPa. The material is then removed from the dye and stored in a sealed glass bottle. 1.5.3.2. Sample Preparation If necessary, organic binder can be removed by placing the fibre inside a furnace at 450 °C for about one hour. Cone and quarter to subdivide the sample (this should be done inside a dust cupboard). Using a spatula, add a small amount (< 0,5 g) of sample to 100 ml of freshly distilled water that has been filtered through a 0,2 μm membrane filter (alternative sources of ultra pure water may be used if they are shown to be satisfactory). Disperse thoroughly by the use of an ultrasonic probe operated at 100 W power and tuned so that cavitation occurs. (If a probe is not available use the following method: repeatedly shake and invert for 30 seconds; ultrasonic in a bench top ultrasonic bath for five minutes; then repeatedly shake and invert for a further 30 seconds.) Immediately after dispersion of the fibre, remove a number of aliquots (e.g. three aliquots of 3, 6 and 10 ml) using a wide-mouthed pipette (2-5 ml capacity). Vacuum filter each aliquot through a 0,2 μm polycarbonate filter supported by a 5 µm pore MEC backing filter, using a 25 mm glass filter funnel with a cylindrical reservoir. Approximately 5 ml of filtered distilled water should be placed into the funnel and the aliquot slowly pipetted into the water holding the pipette tip below the meniscus. The pipette and the reservoir must be flushed thoroughly after pipetting, as thin fibres have a tendency to be located more on the surface. Carefully remove the filter and separate it from the backing filter before placing it in a container to dry. Cut a quarter or half filter section of the filtered deposit with a type 24 scalpel blade using a rocking action. Carefully attach the cut section to a SEM stub using a sticky carbon tab or carbon glue. Silver dag should be applied in at least three positions to improve the electrical contact at the edges of the filter and the stub. When the glue/silver dag is dry, sputter coat approximately 50 nm of gold or gold/palladium onto the surface of the deposit. 1.5.3.3. SEM calibration and operation 1.5.3.3.1. Calibration The SEM calibration should be checked at least once a week (ideally once a day) using a certified calibration grid. The calibration should be checked against a certified standard and if the measured value (SEM) is not within ±2 % of the certified value, then the SEM calibration must be adjusted and re-checked. The SEM should be capable of resolving at least a minimum visible diameter of 0,2 µm, using a real sample matrix, at a magnification of × 2 000. 1.5.3.3.2. Operation The SEM should be operated at 10 000 magnification (2) using conditions that give good resolution with an acceptable image at slow scan rates of, for example, 5 seconds per frame. Although the operational requirements of different SEMs may vary, generally to obtain the best visibility and resolution, with relatively low atomic weight materials, accelerating voltages of 5-10 keV should be used with a small spot size setting and short working distance. As a linear traverse is being conducted, then a 0° tilt should be used to minimise re-focussing or, if the SEM has a eucentric stage, the eucentric working distance should be used. Lower magnification may be used if the material does not contain small (diameter) fibres and the fibre diameters are large (> 5 μm). 1.5.3.4. Sizing 1.5.3.4.1. Low magnification examination to assess the sample Initially the sample should be examined at low magnification to look for evidence of clumping of large fibres and to assess the fibre density. In the event of excessive clumping it is recommended that a new sample is prepared. For statistical accuracy it is necessary to measure a minimum number of fibres and high fibre density may seem desirable as examining empty fields is time consuming and does not contribute to the analysis. However, if the filter is overloaded, it becomes difficult to measure all the measurable fibres and, because small fibres may be obscured by larger ones, they may be missed. Bias towards over estimating the LWGMD may result from fibre densities in excess of 150 fibres per millimetre of linear traverse. On the other hand, low fibre concentrations will increase the time of analysis and it is often cost effective to prepare a sample with a fibre density closer to the optimum than to persist with counts on low concentration filters. The optimum fibre density should give an average of about one or two countable fibre per fields of view at 5 000 magnification. Nevertheless the optimum density will depend on the size (diameter) of the fibres, so it is necessary that the operator uses some expert judgement in order to decide whether the fibre density is close to optimal or not. 1.5.3.4.2. Length weighting of the fibre diameters Only those fibres that touch (or cross) an (infinitely) thin line drawn on the screen of the SEM are counted. For this reason a horizontal (or vertical) line is drawn across the centre of the screen. Alternatively a single point is placed at the centre of the screen and a continuous scan in one direction across the filter is initiated. Each fibre of aspect ratio grater than 3:1 touching or crossing this point has its diameter measured and recorded. 1.5.3.4.3. Fibre sizing It is recommended that a minimum of 300 fibres are measured. Each fibre is measured only once at the point of intersection with the line or point drawn on the image (or close to the point of intersection if the fibre edges are obscured). If fibres with non-uniform cross sections are encountered, a measurement representing the average diameter of the fibre should be used. Care should be taken in defining the edge and measuring the shortest distance between the fibre edges. Sizing may be done on line, or off-line on stored images or photographs. Semi-automated image measurement systems that download data directly into a spreadsheet are recommended, as they save time, eliminate transcription errors and calculations can be automated. The ends of long fibres should be checked at low magnification to ensure that they do not curl back into the measurement field of view and are only measured once. 2. DATA 2.1. TREATMENT OF RESULTS Fibre diameters do not usually have a normal distribution. However, by performing a log transformation it is possible to obtain a distribution that approximates to normal. Calculate the arithmetic mean (mean lnD) and the standard deviation (SDlnD) of the log to base e values (lnD) of the n fibre diameters (D). (1) (2) The standard deviation is divided by the square root of the number of measurements (n) to obtain the standard error (SElnD). (3) Subtract two times the standard error from the mean and calculate the exponential of this value (mean minus two standard errors) to give the geometric mean minus two geometric standard errors. (4) 3. REPORTING TEST REPORT The test report should include at least the following information: — The value of LWGMD-2SE. — Any deviations and particularly those which may have an effect on the precision or accuracy of the results with appropriate justifications. 4. REFERENCES 1. B. Tylee SOP MF 240. Health and Safety Executive, February 1999. 2. G. Burdett and G. Revell. Development of a standard method to measure the length-weigthed geometric mean fibre diameter: Results of the Second inter-laboratory exchange. IR/L/MF/94/07. Project R42.75 HPD. Health and Safety Executive, Research and Laboratory Services Division, 1994. (1) This magnification value is indicated for 3 µm fibres, for 6 µm fibres a magnification of × 5 000 may be more suitable. (2) For 3 μm fibres, see previous note.

ANNEX III B.46. IN VITRO SKIN IRRITATION: RECONSTRUCTED HUMAN EPIDERMIS MODEL TEST 1. METHOD 1.1. INTRODUCTION Skin irritation refers to the production of reversible damage to the skin following the application of a test substance for up to 4 hours [as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Chemicals (GHS)](1). This Test Method provides an in vitro procedure that, depending on information requirements, may allow determining the skin irritancy of substances as a stand-alone replacement test within a testing strategy, in a weight of evidence approach (2). The assessment of skin irritation has typically involved the use of laboratory animals (See Method B.4)(3). In relation to animal welfare concerns, method B.4 allows for the determination of skin corrosion/irritation by applying a sequential testing strategy, using validated in vitro and ex vivo methods, thus avoiding pain and suffering of animals. Three validated in vitro Test Methods or Test Guidelines, B.40, B.40bis and TG 435 (4, 5, 6), are useful for the corrosivity part of the sequential testing strategy of B.4. This Test Method is based on reconstructed human epidermis models, which in their overall design (the use of human derived epidermis keratinocytes as cell source, representative tissue and cytoarchitecture) closely mimic the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis. The procedure described under this Test Method allows the hazard identification of irritant substances in accordance with UN GHS category 2. This Test Method also includes a set of performance standards for the assessment of similar and modified reconstructed human epidermis based test methods (7). Prevalidation, optimisation and validation studies have been completed for two in vitro test methods (8, 9, 10, 11, 12, 13, 14, 15, 16, 17), commercially available as EpiSkin™ and EpiDerm™, using reconstructed human epidermis models. These references were based on R 38. Certain aspects of recalculation for the purposes of GHS are addressed in reference 25. Methods with a performance equivalent to EpiSkin™ (validated reference method 1) are recommended as a stand alone replacement test method for the rabbit in vivo test for classifying GHS category 2 irritant substances. Methods with a performance equivalent to EpiDerm™ (validated reference method 2) are only recommended as a screen test method, or as part of a sequential testing strategy in a weight of evidence approach, for classifying GHS category 2 irritant substances. Before a proposed in vitro reconstructed human epidermis model test for skin irritation can be used for regulatory purposes, its reliability, relevance (accuracy), and limitations for its proposed use should be determined to ensure that it is comparable to that of the validated reference method 1, in accordance with the performance standards set out in this Test Method (Appendix). Two other in vitro reconstructed human epidermis test methods, have been validated in accordance with the requirements under this Test Method, and show similar results as the validated reference method 1 (18). These are the modified EpiDerm™ test method (modified reference method 2) and the SkinEthic RHE™ test method (me-too method 1). 1.2. DEFINITIONS The following definitions are applied within this Test Method: Accuracy: The closeness of agreement between test method results and accepted reference values. It is a measure of test method performance and one aspect of relevance. The term is often used interchangeably with ‘concordance’ to mean the proportion of correct outcomes of a test method. Batch control substance: Benchmark substance producing a mid-range cell viability response of the tissue. Cell viability: Parameter measuring total activity of a cell population e.g. as ability of cellular mitochondrial dehydrogenases to reduce the vital dye MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue;), which, depending on the endpoint measured and the test design used, correlates with the total number and/or vitality of living cells. ET50 : The exposure time required to reduce cell viability by 50 % upon application of the marker substance at a specified, fixed concentration, see also IC50. False negative rate: The proportion of all positive substances falsely identified by a test method as negative. It is one indicator of test method performance. False positive rate: The proportion of all negative (non-active) substances that are falsely identified as positive. It is one indicator of test method performance. Infinite dose: Amount of test substance applied to the skin exceeding the amount required to completely and uniformly cover the skin surface. GHS (Globally Harmonized System of Classification and Labelling of Chemicals): A system proposing the classification of substances and mixtures according to standardised types and levels of physical, health and environmental hazards, and addressing corresponding communication elements, such as pictograms, signal words, hazard statements, precautionary statements and safety data sheets, so that to convey information on their adverse effects with a view to protect people (including employers, workers, transporters, consumers and emergency responders) and the environment (1) and implemented in the EU in Regulation (EC) No 1272/2008. IC50 : The concentration at which a marker substance reduces the viability of the tissues by 50 % (IC50) after a fixed exposure time, see also ET50. Performance standards: Standards, based on a validated reference method, that provide a basis for evaluating the comparability of a proposed test method that is mechanistically and functionally similar. Included are (I) essential test method components; (II) a minimum list of reference substances selected from among the substances used to demonstrate the acceptable performance of the validated reference method; and (III) the comparable levels of accuracy and reliability, based on what was obtained for the validated reference method, that the proposed test method should demonstrate when evaluated using the minimum list of reference substances. Reliability: Measures of the extent that a test method can be performed reproducibly within and between laboratories over time, when performed using the same protocol. It is assessed by calculating intra- and inter-laboratory reproducibility. Sensitivity: The proportion of all positive/active substances that are correctly classified by the test. It is a measure of accuracy for a test method that produces categorical results, and is an important consideration in assessing the relevance of a test method. Specificity: The proportion of all negative/inactive substances that are correctly classified by the test. It is a measure of accuracy for a test method that produces categorical results and is an important consideration in assessing the relevance of a test method. Skin irritation: The production of reversible damage to the skin following the application of a test substance for up to 4 hours. Skin irritation is a locally arising, non-immunogenic reaction, which appears shortly after stimulation (24). Its main characteristic is its reversible process involving inflammatory reactions and most of the clinical characteristic signs of irritation (erythema, oedema, itching and pain) related to an inflammatory process. 1.3. SCOPE AND LIMITATIONS A limitation of the reconstructed human epidermis tests falling under this Test Method is that they only classify substances as skin irritants according to UN GHS category 2. As they do not allow the classification of substances to the optional category 3 as defined in the UN GHS, all remaining substances will not be classified (no category). Depending on regulatory needs and possible future inclusion of new endpoints, improvements or development of new me-too tests, this Test Method may have to be revised. This Test method allows the hazard identification of irritant mono-constituent substances (19), but it does not provide adequate information on skin corrosion. Gases and aerosols cannot be tested, while mixtures have not been assessed yet in a validation study. 1.4. PRINCIPLE OF THE TEST The test substance is applied topically to a three-dimensional reconstructed human epidermis model, comprised of normal, human-derived epidermal keratinocytes, which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organised basal, spinous and granular layers, and a multilayered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The principle of the reconstructed human epidermis model test is based on the premise that irritant substances are able to penetrate the stratum corneum by diffusion and are cytotoxic to the cells in the underlying layers. Cell viability is measured by dehydrogenase conversion of the vital dye MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; EINECS number 206-069-5, CAS number 298-93-1)], into a blue formazan salt that is quantitatively measured after extraction from tissues (20). Irritant substances are identified by their ability to decrease cell viability below defined threshold levels (i.e. ≤ 50 %, for UN GHS category 2 irritants). Substances that produce cell viabilities above the defined threshold level, will not be classified (i.e. > 50 %, no category). The reconstructed human epidermis model systems may be used to test solids, liquids, semi-solids and waxes. The liquids may be aqueous or non-aqueous; solids may be soluble or insoluble in water. Whenever possible, solids should be tested as a fine powder. Since 58 carefully selected substances, representing a wide spectrum of chemical classes, were included in the validation of the reconstructed human epidermis model test systems, the methods are expected to be generally applicable across chemical classes (16). The validation includes 13 GHS Cat. 2 irritants. It should be noted that non-corrosive acids, bases, salts and other inorganic substances were not included in the validation and some known classes of organic irritants such as hydroperoxides, phenols and surfactants were not included or were only included to a limited extent. 1.5. DEMONSTRATION OF PROFICIENCY Prior to routine use of a validated method that adheres to this Test Method, laboratories may wish to demonstrate technical proficiency, using the ten substances recommended in Table 1. Under this Test Method, the UN GHS optional category 3 is considered as no category. For novel similar (me-too) test methods developed under this Test Method that are structurally and functionally similar to the validated reference methods or for modifications of validated methods, the performance standards described in the Appendix to this Test Method should be used to demonstrate comparable reliability and accuracy of the new test method prior to its use for regulatory testing. Table 1 Proficiency Substances which are a subset of the Reference Substances listed in the Appendix Substance CAS Number In vivo score Physical state GHS category naphthalene acetic acid 86-87-3 0 S No Cat. isopropanol 67-63-0 0,3 L No Cat. methyl stearate 112-61-8 1 S No Cat. heptyl butyrate 5870-93-9 1,7 L Optional Cat. 3 hexyl salicylate 6259-76-3 2 L Optional Cat. 3 cyclamen aldehyde 103-95-7 2,3 L Cat. 2 1-bromohexane 111-25-1 2,7 L Cat. 2 butyl methacrylate 97-88-1 3 L Cat. 2 1-methyl-3-phenyl-1-piperazine 5271-27-2 3,3 S Cat. 2 Heptanal 111-71-7 4 L Cat. 2 1.6. DESCRIPTION OF THE METHOD The following is a description of the components and procedures of a reconstructed human epidermis model test for skin irritation assessment. A reconstructed human epidermis model can be constructed, prepared or obtained commercially (e.g. EpiSkin™, EpiDerm™ and SkinEthic RHE™). Standard test method protocols for EpiSkin™, EpiDerm™ and SkinEthic RHE™ can be obtained at [http://ecvam.jrc.ec.europa.eu](21, 22, 23). Testing should be performed according to the following: 1.6.1. Reconstructed Human Epidermis Model Components 1.6.1.1. General model conditions Normal human keratinocytes should be used to construct the epithelium. Multiple layers of viable epithelial cells (basal layer, stratum spinosum, stratum granulosum) should be present under a functional stratum corneum. Stratum corneum should be multilayered containing the essential lipid profile to produce a functional barrier with robustness to resist rapid penetration of cytotoxic marker substances, e.g. sodium dodecyl sulphate (SDS) or Triton X-100. The barrier function may be assessed either by determination of the concentration at which a marker substance reduces the viability of the tissues by 50 % (IC50) after a fixed exposure time, or by determination of the exposure time required to reduce cell viability by 50 % (ET50) upon application of the marker substance at a specified, fixed concentration. The containment properties of the model should prevent the passage of material around the stratum corneum to the viable tissue, which would lead to poor modelling of skin exposure. The skin model should be free of contamination by bacteria, viruses, mycoplasma, or fungi. 1.6.1.2. Functional model conditions 1.6.1.2.1. Viability The preferred assay for determining the magnitude of viability is the MTT (20). The optical density (OD) of the extracted (solubilised) dye from the tissue treated with the negative control (NC) should be at least 20 fold greater than the OD of the extraction solvent alone. It should be documented that the tissue treated with NC is stable in culture (provide similar viability measurements) for the duration of the test exposure period. 1.6.1.2.2. Barrier function The stratum corneum and its lipid composition should be sufficient to resist the rapid penetration of cytotoxic marker substances, e.g. SDS or Triton X-100, as estimated by IC50 or ET50. 1.6.1.2.3. Morphology Histological examination of the reconstructed skin/epidermis should be performed by appropriately qualified personnel demonstrating human skin/epidermis-like structure (including multilayered stratum corneum). 1.6.1.2.4. Reproducibility The results of the method using a specific model should demonstrate reproducibility over time, preferably by an appropriate batch control (benchmark) substance (see Appendix). 1.6.1.2.5. Quality controls (QC) of the model Each batch of the epidermal model used should meet defined production release criteria, among which those for viability (paragraph 1.6.1.2.1) and for barrier function (paragraph 1.6.1.2.2) are the most relevant. An acceptability range (upper and lower limit) for the IC50 or the ET50 should be established by the skin model supplier (or investigator when using an in-house model). The barrier properties of the tissues should be verified by the laboratory after receipt of the tissues. Only results produced with qualified tissues can be accepted for reliable prediction of irritation effects. As an example, the acceptability ranges for the validated reference methods are given below. Table 2 Examples of QC batch release criteria Lower acceptance limit Mean of acceptance range Upper acceptance limit Validated reference method 1 (18 hours treatment with SDS) IC50 = 1,0 mg/ml IC50 = 2,32 mg/ml IC50 = 3,0 mg/ml Validated reference method 2 (1 % Triton X-100) ET50 = 4,8 hr ET50 = 6,7 hr ET50 = 8,7 hr 1.6.1.3. Application of the Test and Control Substances A sufficient number of tissue replicates should be used for each treatment and for controls (at least three replicates per run). For liquid as well as solid substances, sufficient amount of test substance should be applied to uniformly cover the skin surface while avoiding an infinite dose (see 1.2 Definitions), i.e. a minimum of 25 μL/cm2 or 25 mg/cm2 should be used. For solid substances, the epidermis surface should be moistened with deionised or distilled water before application, to ensure good contact with the skin. Whenever possible, solids should be tested as a fine powder. At the end of the exposure period, the test substance should be carefully washed from the skin surface with aqueous buffer, or 0,9 % NaCl. Depending on the reconstructed human epidermis model used, the exposure period may vary between 15 to 60 minutes, and the incubation temperature between 20 and 37 °C. For details, see the Standard Operating Procedures for the three methods (21, 22, 23). Concurrent NC and positive controls (PC) should be used for each study to demonstrate that viability (NC), barrier function and resulting tissue sensitivity (PC) of the tissues are within a defined historical acceptance range. The suggested PC substance is 5 % aqueous SDS. The suggested NC substances are water or phosphate buffered saline (PBS). 1.6.1.4. Cell Viability Measurements The most important element of the test procedure is that viability measurements are not performed immediately after the exposure to the test substances, but after a sufficiently long post-treatment incubation period of the rinsed tissues in fresh medium. This period allows both for recovery from weakly irritant effects and for appearance of clear cytotoxic effects. During the test optimisation phase (9, 10, 11, 12, 13), a 42 hours post-treatment incubation period proved to be optimal and was therefore used in the validation of the reference test methods. The MTT conversion assay is a quantitative validated method which should be used to measure cell viability. It is compatible with use in a three-dimensional tissue construct. The skin sample is placed in MTT solution of appropriate concentration (e.g. 0,3-1 mg/mL) for 3 hours. The precipitated blue formazan product is then extracted from the tissue using a solvent (e.g. isopropanol, acidic isopropanol), and the concentration of formazan is measured by determining the OD at 570 nm using a bandpass of maximum ±30 nm. Optical properties of the test substance or its chemical action on the MTT may interfere with the assay leading to a false estimate of viability (because the test substance may prevent or reverse the colour generation as well as cause it). This may occur when a specific test substance is not completely removed from the skin by rinsing or when it penetrates the epidermis. If the test substance acts directly on the MTT, is naturally coloured, or becomes coloured during tissue treatment, additional controls should be used to detect and correct for test substance interference with the viability measurement technique. For detailed description of how to test direct MTT reduction, please consult the test method protocol for the validated reference methods (21, 22, 23). Non-specific colour (NSC) due to these interferences should not exceed 30 % of NC (for corrections). If NSC > 30 %, the test substance is considered as incompatible with the test. 1.6.1.5. Assay Acceptability Criteria For each assay using valid batches (see paragraph 1.6.1.2.5), tissues treated with the NC should exhibit OD reflecting the quality of the tissues that followed all shipment and receipt steps and all the irritation protocol process. The OD values of controls should not be below historical established lower boundaries. Similarly, tissues treated with the PC, i.e. 5 % aqueous SDS, should reflect the sensitivity retained by tissues and their ability to respond to an irritant substance in the conditions of each individual assay (e.g. viability ≤ 40 % for the validated reference method 1, and ≤ 20 % for the validated reference method 2). Associated and appropriate measures of variability between tissue replicates should be defined (e.g. if standard deviations are used they should be ≤ 18 %). 2. DATA 2.1. DATA For each treatment, data from individual replicate test samples (e.g. OD values and calculated percentage cell viability data for each test substance, including classification) should be reported in tabular form, including data from repeat experiments as appropriate. In addition means ± standard deviation for each trial should be reported. Observed interactions with MTT reagent and coloured test substances should be reported for each tested substance. 2.2. INTERPRETATION OF RESULTS The OD values obtained with each test sample can be used to calculate the percentage of viability compared to NC, which is set at 100 %. The cut-off value of percentage cell viability distinguishing irritant from non-classified test substances and the statistical procedure(s) used to evaluate the results and identify irritant substances, should be clearly defined and documented, and proven to be appropriate. The cut-off values for the prediction of irritation associated with the validated reference methods is given below: The test substance is considered to be irritant to skin in accordance with UN GHS category 2: (i) if the tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50 %. The test substance is considered to have no category: (ii) if the tissue viability after exposure and post-treatment incubation is more than (>) 50 %. 3. REPORTING 3.1. TEST REPORT The test report should include the following information: Test and Control Substances: — chemical name(s) such as IUPAC or CAS name and CAS number, if known, — purity and composition of the substance (in percentage(s) by weight), — physical-chemical properties relevant to the conduct of the study (e.g. physical state, stability and volatility, pH, water solubility if known), — treatment of the test/control substances prior to testing, if applicable (e.g. warming, grinding), — storage conditions. Justification of the skin model and protocol used. Test Conditions: — cell system used, — calibration information for measuring device, and bandpass used for measuring cell viability (e.g. spectrophotometer), — complete supporting information for the specific skin model used including its performance. This should include, but is not limited to: (i) viability; (ii) barrier function; (iii) morphology; (iv) reproducibility and predictivity; (v) quality controls (QC) of the model; — details of the test procedure used, — test doses used, duration of exposure and post treatment incubation period, — description of any modifications of the test procedure, — reference to historical data of the model. This should include, but is not limited to: (i) acceptability of the QC data with reference to historical batch data; (ii) acceptability of the positive and negative control values with reference to positive and negative control means and ranges, — description of evaluation criteria used including the justification for the selection of the cut-off point(s) for the prediction model. Results: — tabulation of data from individual test samples, — description of other effects observed. Discussion of the results. Conclusions. 4. REFERENCES 1. United Nations (UN) (2007). Globally Harmonized System of Classification and Labelling of Chemicals (GHS), Second revised edition, UN New York and Geneva, 2007. Available at: http://www.unece.org/trans/danger/publi/ghs/ghs_rev02/02files_e.html. 2. REACH: Guidance on Information Requirements and Chemical Safety Assessment. Available at: http://guidance.echa.europa.eu/docs/guidance_document/information_requirements_en.htm?time=1232447649. 3. Test Method B.4. ACUTE TOXICITY; DERMAL IRRITATION/CORROSION. 4. Test Method B.40. IN VITRO SKIN CORROSION: TRANSCUTANEOUS ELECTRICAL RESISTANCE TEST TER. 5. Test Method B.40 BIS. IN VITRO SKIN CORROSION: HUMAN SKIN MODEL TEST. 6. OECD (2006). Test Guideline 435. OECD Guideline for the Testing of Chemicals. In Vitro Membrane Barrier Test Method. Adopted July 19, 2006. Available at: http://www.oecd.org/document/22/0,2340,en_2649_34377_1916054_1_1_1_1,00.html. 7. ECVAM (2009) Performance Standards for applying human skin models to in vitro skin irritation. Available under Download Study Documents, at: http://ecvam.jrc.ec.europa.eu. 8. Fentem, J.H., Briggs, D., Chesné, C., Elliot, G.R., Harbell, J.W., Heylings, J.R., Portes, P., Roguet, R., van de Sandt, J.J.M. & Botham, P. (2001). A prevalidation study on in vitro tests for acute skin irritation. Results and evaluation by the Management Team. Toxicology in Vitro 15, 57-93. 9. Portes, P., Grandidier, M.H., Cohen, C. & Roguet, R.(2002). Refinement of the EPISKIN protocol for the assessment of acute skin irritation of chemicals: follow-up to the ECVAM prevalidation study. Toxicology in Vitro 16, 765-770. 10. Kandárová, H., Liebsch, M., Genschow, E., Gerner, I., Traue, D., Slawik, B. & Spielmann, H. (2004). Optimisation of the EpiDerm test protocol for the upcoming ECVAM validation study on in vitro skin irritation tests. ALTEX 21, 107-114. 11. Kandárová, H., Liebsch, M., Gerner, I., Schmidt, E., Genschow, E., Traue, D. & Spielmann H. (2005) The EpiDerm Test Protocol fort the Upcoming ECVAM Validation Study on In Vitro Skin Irritation Tests — An Assessment of the Performance of the Optimised Test. ATLA 33, 351-367. 12. Cotovio, J., Grandidier, M.-H., Portes, P., Roguet, R. & G. Rubinsteen. (2005). The In Vitro Acute Skin Irritation of Chemicals: Optimisation of the EPISKIN Prediction Model within the Framework of the ECVAM Validation Process. ATLA 33, 329-249. 13. Zuang, V., Balls, M., Botham, P.A., Coquette, A., Corsini, E., Curren, R.D., Elliot, G.R., Fentem, J.H., Heylings, J.R., Liebsch, M., Medina, J., Roguet, R., van De Sandt, J.J.M., Wiemann, C. & Worth, A.(2002). Follow-up to the ECVAM prevalidation study on in vitro tests for acute skin irritation. ECVAM Skin Irritation Task Force Report 2. ATLA 30,109-129. 14. Spielmann, H., Hoffmann, S., Liebsch, M., Botham, P., Fentem, J., Eskes, C., Roguet, R., Cotovió, J., Cole, T., Worth, A., Heylings, J., Jones, P., Robles, C., Kandárová, H., Gamer, A., Remmele, M., Curren, R., Raabe, H., Cockshott, A., Gerner, I. and Zuang, V. (2007) The ECVAM International Validation Study on In Vitro Tests for Acute Skin Irritation: Report on the Validity of the EPISKIN and EpiDerm Assays and on the Skin Integrity Function Test. ATLA 35, 559-601. 15. Hoffmann, S. (2006). ECVAM Skin Irritation Validation Study Phase II: Analysis of the Primary Endpoint MTT and the Secondary Endpoint IL1-α. 135 pp. + annexes. Available under Download Study Documents, at: http://ecvam.jrc.ec.europa.eu. 16. Eskes, C., Cole, T., Hoffmann, S., Worth, A., Cockshott, A., Gerner, I. & Zuang. V (2007) ECVAM International Validation Study on In Vitro Tests for Acute Skin Irritation: Selection of Test Chemicals. ATLA 35, 603-619. 17. J. Cotovio, M.-H. Grandidier, D. Lelièvre, R. Roguet, E. Tinois-Tessonneaud, J. Leclaire (2007). In vitro acute skin irritancy of chemicals using the validated EPISKIN model in a tiered strategy — Results and performances with 184 cosmetic ingredients, AATEX, Special Issue-proceedings from WC6. Vol.14, 351-358. 18. ESAC statement on updated EpiDerm and similar SkinEthic assays. 5 November 2008. 19. EC (2006). Regulation (EC) No 1907/2006 of the European Parliament and of the Council of 18 December 2006 concerning the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), establishing a European Chemicals Agency, amending Directive 1999/45/EC and repealing Council Regulation (EEC) No 793/93 and Commission Regulation (EC) No 1488/94 as well as Council Directive 76/769/EEC and Commission Directives 91/155/EEC, 93/67/EEC, 93/105/EC and 2000/21/EC. Official Journal of the European Union L 396 of 30 December 2006, p. 1. OPOCE, Luxembourg. 20. Mosman, T. (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of Immunological Methods 65, 55-63. 21. EpiSkin™ SOP, Version 1.6 (January 2005). Validation of the EpiSkin Skin Irritation Test — 42 Hours Assay For The Prediction of Acute Skin Irritation of Chemicals. Available under Download Study Documents, at: http://ecvam.jrc.ec.europa.eu. 22. EpiDerm™ SOP, Version 5.0 (October 2004). Draft Standard Operating Procedure. In Vitro Skin Irritation Test: Human Skin Model. Model: EpiDerm™- 200. Available under Download Study Documents, at: http://ecvam.jrc.ec.europa.eu. 23. SkinEthic RHE™ SOP. Will be available under Download Study Documents, at: http://ecvam.jrc.ec.europa.eu. 24. Harvell, J.D., Lammintausta, K., Maibach H.I. (1995) Irritant contact dermatitis IN: Guin J.D. Practical Contact Dermatitis Mc Graw-Hill New York, pp 7-18 25. Griesinger C, Barroso J & Zuang V: ECVAM background document on the recent adaptations of the ECVAM performance standards for in vitro skin irritation testing in the context of the drafting process of an EU test method and an OECD draft test guideline. Ispra, November 13, 2008. Appendix Assessment of the performance characteristics of proposed in vitro reconstructed human epidermis models for skin irritation INTRODUCTION Procedures proposed for use under this Test Method should be evaluated to determine their reliability and accuracy using substances representing the full range of Draize irritancy scores. When evaluated using the 20 recommended reference substances (Table 2), the proposed procedure should have reliability and accuracy values which are comparable to that of the validated reference method 1 (Table 3) (1). The accuracy and reliability standards that should be achieved are provided under II and III below. Non-classified and classified (UN GHS category 2) substances, representing relevant chemical classes are included, so that the reliability and performance (sensitivity, specificity, false negative rates, and false positive rates and accuracy) of the proposed test method can be compared to that of the validated reference method 1. The reliability of the test method, as well as its ability to correctly identify UN GHS category 2 irritant substances, should be determined prior to its use for testing new substances. PERFORMANCE STANDARDS The Performance Standards comprise the following three elements I) Essential Test Method Components, II) Reference Substances and III) Defined Accuracy and Reliability Values (2). These Performance Standards are based on the Performance Standards defined after the completion of the ECVAM skin irritation validation study (3). (I) Essential Test Method Components General model conditions Normal human keratinocytes should be used to construct the epithelium. Multiple layers of viable epithelial cells (basal layer, stratum spinosum, stratum granulosum) should be present under a functional stratum corneum. Stratum corneum should be multilayered containing the essential lipid profile to produce a functional barrier with robustness to resist rapid penetration of cytotoxic marker substances, e.g. SDS or Triton X-100. The barrier function may be assessed either by determination of the concentration at which a marker substance reduces the viability of the tissues by 50 % (IC50) after a fixed exposure time, or by determination of the exposure time required to reduce cell viability by 50 % (ET50) upon application of the marker substance at a specified, fixed concentration. The containment properties of the model should prevent the passage of material around the stratum corneum to the viable tissue, which would lead to poor modelling of skin exposure. The skin model should be free of contamination by bacteria, viruses, mycoplasma, or fungi. Functional model conditions Viability The preferred assay for determining the magnitude of viability is the MTT (4). The OD of the extracted (solubilised) dye from the tissue treated with NC should be at least 20 fold greater than the OD of the extraction solvent alone. It should be documented that the tissue treated with NC is stable in culture (provide similar viability measurements) for the duration of the test exposure period. Barrier function The stratum corneum and its lipid composition should be sufficient to resist the rapid penetration of cytotoxic marker substances, e.g. SDS or Triton X-100, as estimated by IC50 or ET50. Morphology Histological examination of the reconstructed skin/epidermis should be performed by appropriately qualified personnel demonstrating human skin/epidermis-like structure (including multilayered stratum corneum). Reproducibility The results of the method using a specific model should demonstrate reproducibility over time, preferably by an appropriate batch control (benchmark) substance (see definitions in section 1.2). Quality controls (QC) of the model Each batch of the epidermal model used should meet defined production release criteria, among which those for viability and for barrier function are the most relevant. An acceptability range (upper and lower limit) for the IC50 or the ET50 should be established by the skin model supplier (or investigator when using an in-house model). The barrier properties of the tissues should be verified by the laboratory after receipt of the tissues. Only results produced with qualified tissues can be accepted for reliable prediction of irritation effects. As an example, the acceptability ranges for the validated reference methods are given below. Table 1 Examples of QC batch release criteria lower acceptance limit mean of acceptance range upper acceptance limit Validated reference method 1 (18 hours treatment with SDS) IC50 = 1,0 mg/ml IC50 = 2,32 mg/ml IC50 = 3,0 mg/ml Validated reference method 2 (1 % Triton X-100) ET50 = 4,8 hr ET50 = 6,7 hr ET50 = 8,7 hr (II) Reference Substances Reference substances are used to determine if the reliability and accuracy of a proposed novel in vitro reconstructed human epidermis test method, proven to be structurally and functionally sufficiently similar to the validated reference methods, or representing a minor modification of a validated reference method, shows comparable performance to that of the validated reference method 1 (1). The 20 reference substances listed in Table 2 include substances representing different chemical classes of interest, as well as substances in UN GHS category 2. The substances included in this list comprise 10 UN GHS category 2 substances, 3 UN GHS optional category 3 substances and 7 non-categorised substances. Under this Test Method, the optional category 3 is considered as no category. These reference substances represent the minimum number of substances that should be used to evaluate the accuracy and reliability of a proposed reconstructed human epidermis test method for skin irritation. In situations where a listed substance is unavailable, other substances for which adequate in vivo reference data are available could be used. If desired, additional substances representing other chemical classes and for which adequate in vivo reference data are available may be added to the minimum list of reference substances to further evaluate the accuracy of the proposed test method. Table 2 Reference Substances for Determination of Accuracy and Reliability Values for Reconstructed Human Epidermis Skin Irritation Models Substance (1) CAS No EINECS No Physical state In vivo score GHS in vitro cat. GHS in vivo cat. 1-bromo-4-chlorobutane 6940-78-9 230-089-3 L 0 Cat. 2 No Cat. Diethyl phthalate 84-66-2 201-550-6 L 0 No Cat. No Cat. Naphthalene acetic acid 86-87-3 201-705-8 S 0 No Cat. No Cat. Allyl phenoxy-acetate 7493-74-5 231-335-2 L 0,3 No Cat. No Cat. Isopropanol 67-63-0 200-661-7 L 0,3 No Cat. No Cat. 4-methyl-thio-benzaldehyde 3446-89-7 222-365-7 L 1 Cat. 2 No Cat. Methyl stearate 112-61-8 203-990-4 S 1 No Cat. No Cat. Heptyl butyrate 5870-93-9 227-526-5 L 1,7 No Cat. Optional Cat. 3 Hexyl salicylate 6259-76-3 228-408-6 L 2 No Cat. Optional Cat. 3 Tri-isobutyl phosphate 126-71-6 204-798-3 L 2 Cat. 2 Optional Cat. 3 1-decanol 112-30-1 203-956-9 L 2,3 Cat. 2 Cat. 2 Cyclamen aldehyde 103-95-7 203-161-7 L 2,3 Cat. 2 Cat. 2 1-bromohexane 111-25-1 203-850-2 L 2,7 Cat. 2 Cat. 2 2-chloromethyl-3,5-dimethyl-4-methoxypyridine hydrochloride 86604-75-3 434-680-9 S 2,7 Cat. 2 Cat. 2 a-terpineol 98-55-5 202-680-6 L 2,7 Cat. 2 Cat. 2 di-n-propyl disulphide 629-19-6 211-079-8 L 3 No Cat. Cat. 2 Butyl methacrylate 97-88-1 202-615-1 L 3 Cat. 2 Cat. 2 Benzenethiol, 5-(1,1-dimethylethyl)-2-methyl 7340-90-1 438-520-9 L 3,3 Cat. 2 Cat. 2 1-methyl-3-phenyl-1-piperazine 5271-27-2 431-180-2 S 3,3 Cat. 2 Cat. 2 Heptanal 111-71-7 203-898-4 L 4 Cat. 2 Cat. 2 The substances listed in Table 2 provide a representative distribution of the 58 substances used in the ECVAM international skin irritation validation study (1). Their selection is based on the following criteria: — the substances are commercially available, — they are representative of the full range of Draize irritancy scores (from non-irritant to strong irritant), — they have a well-defined chemical structure, — they are representative of the validated method’s reproducibility and predictive capacity as determined in the ECVAM validation study, — they are representative of the chemical functionality used in the validation process, — they are not associated with an extremely toxic profile (e.g. carcinogenic or toxic to the reproductive system) and they are not associated with prohibitive disposal costs. (III) Defined Accuracy and Reliability Values The performance (sensitivity, specificity, false negative rate, false positive rate and accuracy) of the proposed test method should be comparable to that of the validated reference method 1 (Table 3), i.e. sensitivity should be equal or higher (≥) than 80 %, specificity should be equal or higher (≥) than 70 %, and accuracy should be equal or higher (≥) than 75 %. The calculation of the performance should be done using all classifications obtained for the 20 substances in the different participating laboratories. The classification for each substance in each laboratory should be obtained by using the mean value of viability over the different runs performed (minimum three valid runs). Table 3 Performance of the Validated Reference Method 1 (2) Test method No. of Substances Sensitivity Specificity False Negative Rate False Positive rate Accuracy Validated Reference Method 1 (3) 58 87,2 % (4) 71,1 % (5) 12,8 % 29,9 % 74,7 % Validated Reference Method 1 (3) 20 90 % 73,3 % 10 % 26,7 % 81,7 % The reliability of the proposed test method should be comparable to that of the validated reference methods. Within-laboratory reproducibility An assessment of within-laboratory variability should show a concordance of classifications (category 2/no category) obtained in different, independent test runs of the 20 Reference Substances within one single laboratory equal or higher (≥) than 90 %. Between-laboratory reproducibility An assessment of between-laboratory reproducibility is not essential if the proposed test method is to be used in one laboratory only. For methods to be transferred between laboratories, the concordance of classifications (category 2/no category) obtained in different, independent test runs of the 20 Reference Substances between preferentially a minimum of three laboratories should be equal or higher (≥) than 80 %. REFERENCES 1. Spielmann, H., Hoffmann, S., Liebsch, M., Botham, P., Fentem, J., Eskes, C., Roguet, R., Cotovió, J., Cole, T., Worth, A., Heylings, J., Jones, P., Robles, C., Kandárová, H., Gamer, A., Remmele, M., Curren, R., Raabe, H., Cockshott, A., Gerner, I. and Zuang, V. (2007) The ECVAM International Validation Study on In Vitro Tests for Acute Skin Irritation: Report on the Validity of the EPISKIN and EpiDerm Assays and on the Skin Integrity Function Test. ATLA 35, 559-601. 2. OECD (2005) Guidance Document No. 34 on the validation and international acceptance of new or updated test methods for hazard assessment. OECD, Paris. 3. ECVAM (2007) Performance Standards for applying human skin models to in vitro skin irritation. Available under Download Study Documents, at http://ecvam.jrc.ec.europa.eu. Accessed on 27.10.2008. 4. Mosman, T. (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of Immunological Methods 65, 55-63. 5. Eskes, C., Cole, T., Hoffmann, S., Worth, A., Cockshott, A., Gerner, I. & Zuang. V (2007) ECVAM International Validation Study on In Vitro Tests for Acute Skin Irritation: Selection of Test Chemicals. ATLA 35, 603-619. (1) The 20 reference substances comprise a representative selection from the 58 substances which were originally used to validate reference method 1 (EpiSkin™). A complete list of test substances and the criteria for their selection are available (5). (2) Table 3 provides the performance of the validated reference method 1, with regard to its ability to correctly identify irritant substances (UN GHS category 2) and non-classified substances (no category including optional category 3) for the 58 and 20 Reference Substances (Table 2), respectively. (3) EpiSkin™ (4) Based on 13 GHS cat. 2 irritants. (5) Based on 45 GHS cat. 3 irritants or GHS no category chemicals.

ANNEX IV C.3. FRESHWATER ALGAE AND CYANOBACTERIA, GROWTH INHIBITION TEST 1. METHOD This method is equivalent to OECD TG 201 (2006) (1). 1.1. INTRODUCTION Testing Methods are periodically reviewed and updated in the light of scientific progress. Testing Method C.3 needed to be revised to include additional species and to meet the requirements for hazard assessment and classification of chemicals. The revision has been completed on the basis of extensive practical experience, scientific progress in the field of algal toxicity studies, and extensive regulatory use, which has occurred since the original adoption. 1.2. DEFINITIONS The following definitions and abbreviations are used for the purposes of this Testing Method: Biomass: is the dry weight of living matter present in a population expressed in terms of a given volume; e.g. mg algae/litre test solution. Usually ‘biomass’ is defined as a mass, but in this test this word is used to refer to mass per volume. Also in this test, surrogates for biomass, such as cell counts, fluorescence, etc. are typically measured and the use of the term ‘biomass’ thus refers to these surrogate measures as well. Coefficient of variation: is a dimensionless measure of the variability of a parameter, defined as the ratio of the standard deviation to the mean. This can also be expressed as a percentage value. The mean coefficient of variation of average specific growth rate in replicate control cultures should be calculated as follows: 1. Calculate % CV of average specific growth rate out of the daily/section-by-section growth rates for the respective replicate. 2. Calculate the mean value of all the values calculated in point 1 to get the mean coefficient of variation of the daily/section-by-section specific growth rate in replicate control cultures. ECx : is the concentration of the test substance dissolved in the test medium that results in an x % (e.g. 50 %) reduction in growth of the test organism within a stated exposure period (to be mentioned explicitly if deviating from full or normal test duration). To unambiguously denote an EC value deriving from the growth rate or from the yield, the symbols ‘ErC’ and ‘EyC’ are used respectively. Growth medium: is the complete synthetic culture medium in which test algae grow when exposed to the test substance. The test substance will normally be dissolved in the test medium. Growth rate (average specific growth rate): is the logarithmic increase in biomass during the exposure period. Lowest Observed Effect Concentration (LOEC): is the lowest tested concentration at which the substance is observed to have a statistically significant reducing effect on growth (at p < 0,05) when compared with the control, within a given exposure time. However, all test concentrations above the LOEC must have a harmful effect equal to or greater than those observed at the LOEC. When these two conditions cannot be satisfied, a full explanation must be given for how the LOEC (and hence the NOEC) has been selected. No Observed Effect Concentration (NOEC): is the test concentration immediately below the LOEC. Response variable: is a variable for the estimation of toxicity derived from any measured parameters describing biomass by different methods of calculation. For this method, growth rates and yield are response variables derived from measuring biomass directly or any of the surrogates mentioned. Specific growth rate: is a response variable defined as the quotient of the difference of the natural logarithms of a parameter of observation (in this Testing Method, biomass) and the respective time period. Yield: is the value of a measurement variable at the end of the exposure period minus the measurement variable’s value at the start of the exposure period to express biomass increase during the test. 1.3. APPLICABILITY OF THE TEST This Testing Method is most easily applied to water-soluble substances which, under the conditions of the test, are likely to remain in the water. For testing of substances that are volatile, strongly adsorbing, coloured, having a low solubility in water or substances that may affect the availability of nutrients or minerals in the test medium, certain modifications of the described procedure may be required (e.g. closed system, conditioning of the test vessels). Guidance on some appropriate modifications is given in (2), (3) and (4). 1.4. PRINCIPLE OF THE TEST The purpose of this test is to determine the effects of a substance on the growth of freshwater microalgae and/or cyanobacteria. Exponentially growing test organisms are exposed to the test substance in batch cultures over a period of normally 72 hours. In spite of the relatively brief test duration, effects over several generations can be assessed. The system response is the reduction of growth in a series of algal cultures (test units) exposed to various concentrations of a test substance. The response is evaluated as a function of the exposure concentration in comparison with the average growth of replicate, unexposed control cultures. For full expression of the system response to toxic effects (optimal sensitivity), the cultures are allowed unrestricted exponential growth under sufficient nutrient conditions and continuous light for a sufficient period of time to measure reduction of the specific growth rate. Growth and growth inhibition are quantified by measurements of the algal biomass as a function of time. Algal biomass is defined as the dry weight per volume, e.g. mg algae/litre test solution. However, dry weight is difficult to measure and therefore surrogate parameters are used. Of these surrogates, cell counts are most often used. Other surrogate parameters include cell volume, fluorescence, optical density, etc. A conversion factor between the measured surrogate parameter and biomass should be known. The test endpoint is inhibition of growth, expressed as the logarithmic increase in biomass (average specific growth rate) during the exposure period. From the average specific growth rates recorded in a series of test solutions, the concentration bringing about a specified x % inhibition of growth rate (e.g. 50 %) is determined and expressed as the ErCx (e.g. ErC50). For the application of this method within the EU regulatory framework, calculation of results should be based on an average specific growth rate for the reasons described in section 2.2 below. An additional response variable used in this Testing Method is yield, which may be needed to fulfil specific regulatory requirements in some countries. It is defined as biomass at the end of the exposure period minus the biomass at the start of the exposure period. From the yield recorded in a series of test solutions, the concentration bringing about a specified x % inhibition of yield (e.g. 50 %) is calculated and expressed as the EyCx (e.g. EyC50). In addition, the lowest observed effect concentration (LOEC) and the no observed effect concentration (NOEC) may be statistically determined. 1.5. INFORMATION ON THE TEST SUBSTANCE Information on the test substance, which may be useful in establishing the test conditions, includes the structural formula, purity, stability in light, stability under the conditions of the test, light absorption properties, pKa, and results of studies of transformation including biodegradability in water. The water solubility, octanol water partition coefficient (Pow) and the vapour pressure of the test substance should be known, and a validated method for the quantification of the substance in the test solutions with reported recovery efficiency and limit of detection should be available. 1.6. REFERENCE SUBSTANCE Reference substance(s), such as 3,5-dichlorophenol used in the international ring test (4), may be tested as a means of checking the test procedure. Potassium dichromate can also be used as a reference substance for green algae. It is desirable to test a reference substance at least twice a year. 1.7. VALIDITY OF THE TEST For the test to be valid, the following performance criteria should be met: — The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period. This corresponds to a specific growth rate of 0,92 day–1. For the most frequently used species, the growth rate is usually substantially higher (see Appendix 1). This criterion may not be met when species that grow slower than those listed in Appendix 1 are used. In this case, the test period should be extended to obtain at least a 16-fold growth in control cultures, while the growth has to be exponential throughout the test period. The test period may be shortened to at least 48 h to maintain unlimited exponential growth during the test, as long as the minimum multiplication factor of 16 is reached. — The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures (see section 1.2 under ‘coefficient of variation’) must not exceed 35 %. See the second paragraph in section 2.2.1 for the calculation of section-by-section specific growth rate. This criterion applies to the mean value of coefficients of variation calculated for replicate control cultures. — The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7 % in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10 %. 1.8. DESCRIPTION OF THE METHOD 1.8.1. Apparatus Test vessels and other apparatus, which will come into contact with the test solutions should be made entirely of glass or other chemically inert material. The items should be thoroughly washed to ensure that no organic or inorganic contaminants may interfere with the algal growth or composition of the test solutions. The test vessels will normally be glass flasks of dimensions that allow a sufficient volume of culture for measurements during the test and a sufficient mass transfer of CO2 from the atmosphere (see the second paragraph in section 1.8.9). Note that the liquid volume must be sufficient for analytical determinations (see the fifth paragraph in section 1.8.11). In addition, some or all of the following equipment will be required: — Culturing apparatus: a cabinet or chamber is recommended, in which the chosen incubation temperature can be maintained at ±2 °C. — Light measurement instruments: it is important to note that the method of measurement of light intensity, and in particular the type of receptor (collector), wil