Council Regulation (EC) No 440/2008 of 30 May 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)

• (4) OECD (2000). OECD Draft Guideline for the Testing of Chemicals: 122 Partition Coefficient (n-Octanol/Water): pH-Metric Method for Ionisable Substances. Paris.

• (5) Tolls J (2002). Partition Coefficient 1-Octanol/Water (Pow) Slow-Stirring Method for Highly Hydrophobic Chemicals, Validation Report. RIVM contract-Nrs 602730 M/602700/01.

• (6) Boethling RS, Mackay D (eds.) (2000). Handbook of property estimation methods for chemicals. Lewis Publishers Boca Raton, FL, USA.

• (7) Schwarzenbach RP, Gschwend PM, Imboden DM (1993). Environmental Organic Chemistry. Wiley, New York, NY.

• (8) Arnold CG, Widenhaupt A, David MM, Müller SR, Haderlein SB, Schwarzenbach RP (1997). Aqueous speciation and 1-octanol-water partitioning of tributyl- and triphenyltin: effect of pH and ion composition. Environ. Sci. Technol. 31: 2596-2602.

• (9) OECD (1981) OECD Guidelines for the Testing of Chemicals: 112 Dissociation Constants in Water. Paris.

• (10) Chapter A.6 of this Annex, Water Solubility.

• (11) Chapter C.7 of this Annex, Degradation – Abiotic Degradation Hydrolysis as a Function of pH.

• (12) Chapter C.4 — Part II – VII (Method A to F) of this Annex, Determination of ‘Ready’ Biodegradability.

• (13) Chapter A.4 of this Annex, Vapour Pressure.

• (14) Pinsuwan S, Li A and Yalkowsky S.H. (1995). Correlation of octanol/water solubility ratios and partition coefficients, J. Chem. Eng. Data. 40: 623-626.

• (15) Lyman WJ (1990). Solubility in water. In: Handbook of Chemical Property Estimation Methods: Environmental Behavior of Organic Compounds, Lyman WJ, Reehl WF, Rosenblatt DH, Eds. American Chemical Society, Washington, DC, 2-1 to 2-52.

• (16) Leo A, Weininger D (1989). Medchem Software Manual. Daylight Chemical Information Systems, Irvine, CA.

• (17) Meylan W (1993). SRC-LOGKOW for Windows. SRC, Syracuse, N.Y.

• (18) Compudrug L (1992). ProLogP. Compudrug, Ltd, Budapest.

• (19) ACD. ACD logP; Advanced Chemistry Development: Toronto, Ontario M5H 3V9, Canada, 2001.

• (20) Lyman WJ (1990). Octanol/water partition coefficient. In Lyman WJ, Reehl WF, Rosenblatt DH, eds, Handbook of chemical property estimation, American Chemical Society, Washington, D.C.

• (21) Rekker RF, de Kort HM (1979). The hydrophobic fragmental constant: An extension to a 1 000 data point set. Eur. J. Med. Chem. Chim. Ther. 14: 479-488.

• (22) Jübermann O (1958). Houben-Weyl, ed, Methoden der Organischen Chemie: 386-390.

Appendix 1

Spreadsheet for computation of minimum volumes of water required for detection of test substances of different log POW values in aqueous phase

Assumptions:

• — Maximum volume of individual aliquots = 10 % of total volume; 5 aliquots = 50 % of total volume.

• — Image omitted; see official source. In case of lower concentrations, larger volumes would be required.

• — Volume used for LOD determination = 100 ml.

• — log Pow vs. log Sw and log Pow vs. SR (Soct/Sw) are reasonable representations of relationships for test substances.

Estimation of Sw

log Pow	Equation	log Sw	Sw (mg/l)
4	Image omitted; see official source	0,496	3,133E+00
4,5	Image omitted; see official source	0,035	1,084E+00
5	Image omitted; see official source	–0,426	3,750E-01
5,5	Image omitted; see official source	–0,887	1,297E-01
6	Image omitted; see official source	–1,348	4,487E-02
6,5	Image omitted; see official source	––1,809	1,552E-02
7	Image omitted; see official source	–2,270	5,370E-03
7,5	Image omitted; see official source	–2,731	1,858E-03
8	Image omitted; see official source	–3,192	6,427E-04

Estimation of Soct

log Pow	Equation	Soct (mg/l)
4	Image omitted; see official source	3,763E+04
4,5	Image omitted; see official source	4,816E+04
5	Image omitted; see official source	6,165E+04
5,5	Image omitted; see official source	7,890E+04
6	Image omitted; see official source	1,010E+05
6,5	Image omitted; see official source	1,293E+05
7	Image omitted; see official source	1,654E+05
7,5	Image omitted; see official source	2,117E+05
8	Image omitted; see official source	2,710E+05
Total Mass test substance (mg)	Massoct/Masswater	MassH2O (mg)
---	---	---
1 319	526	2,5017
1 686	1 664	1,0127
2 158	5 263	0,4099
2 762	16 644	0,1659
3 535	52 632	0,0672
4 524	1664 36	0,0272
5 790	5263 16	0,0110
7 411	1 664 357	0,0045
9 486	5 263 158	0,0018

Computation of volumes

Minimum volume required for H2O phase at each LOD concentration

log Kow	LOD (micrograms/l)→	0,001	0,01	0,10	1,00	10
4		0,04	0,38	3,80	38	380
4,5		0,09	0,94	9,38	94	938
5		0,23	2,32	23,18	232	2 318
5,5		0,57	5,73	57,26	573	5 726
6		1,41	14,15	141	1 415	14 146
6,5		3,50	34,95	350	3 495	34 950
7		8,64	86,35	864	8 635	86 351
7,5		21,33	213	2 133	21 335	213 346
8		52,71	527	5 271	52 711	527 111
Volume used for LOD (l)	0,1

Key to Computations

Represents < 10 % of total volume of aqueous phase, 1 litre equilibration vessel.

Represents < 10 % of total volume of aqueous phase, 2 litre equilibration vessel.

Represents < 10 % of total volume of aqueous phase, 5 litre equilibration vessel.

Represents < 10 % of total volume of aqueous phase, 10 litre equilibration vessel.

Exceeds 10 % of even the 10 liter equilibration vessel.

Overview of volumes required, as a function of water solubility and Log Pow

Minimum volume required for H2O phase at each LOD concentration (ml)

log Pow	Sw (mg/l)	LOD (micrograms/l)→	0,001	0,01	0,10	1,00	10
4	10		0,01	0,12	1,19	11,90	118,99
	5		0,02	0,24	2,38	23,80	237,97
	3		0,04	0,40	3,97	39,66	396,62
	1		0,12	1,19	11,90	118,99	1 189,86
4,5	5		0,02	0,20	2,03	20,34	203,37
	2		0,05	0,51	5,08	50,84	508,42
	1		0,10	1,02	10,17	101,68	1 016,83
	0,5		0,20	2,03	20,34	203,37	2 033,67
5	1		0,09	0,87	8,69	86,90	869,01
	0,5		0,17	1,74	17,38	173,80	1 738,02
	0,375		0,23	2,32	23,18	231,75	2 317,53
	0,2		0,43	4,35	43,45	434,51	4 345,05
5,5	0,4		0,19	1,86	18,57	185,68	1 856,79
	0,2		0,37	3,71	37,14	371,36	3 713,59
	0,1		0,74	7,43	74,27	742,72	7 427,17
	0,05		1,49	14,85	148,54	1 485,43	14 854,35
6	0,1		0,63	6,35	63,48	634,80	6 347,95
	0,05		1,27	12,70	126,96	1 269,59	12 695,91
	0,025		2,54	25,39	253,92	2 539,18	25 391,82
	0,0125		5,08	50,78	507,84	5 078,36	50 783,64
6,5	0,025		2,17	21,70	217,02	2 170,25	21 702,46
	0,0125		4,34	43,40	434,05	4 340,49	43 404,93
	0,006		9,04	90,43	904,27	9 042,69	90 426,93
	0,003		18,09	180,85	1 808,54	18 085,39	180 853,86
7	0,006		7,73	77,29	772,89	7 728,85	77 288,50
	0,003		15,46	154,58	1 545,77	15 457,70	154 577,01
	0,0015		23,19	231,87	2 318,66	23 186,55	231 865,51
	0,001		46,37	463,73	4 637,31	46 373,10	463 731,03
7,5	0,002		19,82	198,18	1 981,77	19 817,73	198 177,33
	0,001		39,64	396,35	3 963,55	39 635,47	396 354,66
	0,0005		79,27	792,71	7 927,09	79 270,93	792 709,32
	0,00025		158,54	1 585,42	15 854,19	158 541,86	1 585 418,63
8	0,001		33,88	338,77	3 387,68	33 876,77	338 767,72
	0,0005		67,75	677,54	6 775,35	67 753,54	677 535,44
	0,00025		135,51	1 355,07	13 550,71	135 507,09	1 355 070,89
	0,000125		271,01	2 710,14	27 101,42	271 014,18	2 710 141,77
Volume used for LOD (l)	Volume used for LOD (l)	0,1

Appendix 2

An example of glass-jacketed test vessel for the slow-stirring experiment for determination of POW

Image omitted; see official source

A.24.   PARTITION COEFFICIENT (N-OCTANOL/WATER), HIGH PERFORMANCELIQUID CHROMATOGRAPHY (HPLC) METHOD

INTRODUCTION

This test method is equivalent to OECD test guideline (TG) 117 (2004)

• 1. The partition coefficient (P) is defined as the ratio of the equilibrium concentrations of a dissolved substance in a two-phase system consisting of two largely immiscible solvents. In the case of n-octanol and water, Image omitted; see official source The partition coefficient being the quotient of two concentrations, is dimensionless and is usually given in the form of its logarithm to base ten.

• 1. Pow is a key parameter in studies of the environmental fate of chemical substances. A highly-significant relationship between the Pow of non-ionised form of substances and their bioaccumulation in fish has been shown. It has also been shown that Pow is a useful parameter in the prediction of adsorption on soil and sediments and for establishing quantitative structure-activity relationships for a wide range of biological effects.

• 1. The original proposal for this test method was based on an article by C.V. Eadsforth and P. Moser (1). The development of the test method and an OECD inter-laboratory comparison test were coordinated by the Umweltbundesamt of the Federal Republic of Germany during 1986 (2).

INITIAL CONSIDERATIONS

• 4.log Pow values in the range – 2 to 4 (occasionally up to 5 and more) ([^E0010]) can be experimentally determined by the Shake-Flask method (Chapter A.8 of this Annex, OECD Test Guideline 107). The HPLC method covers log Pow in the range of 0 to 6 (1)(2)(3)(4)(5). This method may require an estimation of Pow to assign suitable reference substances and support any conclusions drawn from the data generated by the test. Calculation methods are briefly discussed in the Appendix to this test method. The HPLC operation mode is isocratic.

• 5.The Pow values depend on the environmental conditions such as temperature, pH, ionic strength etc, and these should be defined in the experiment for the correct interpretation of Pow data. For ionisable substances, another method (e.g. draft OECD guideline on pH metric method for ionised substances (6)) may become available and could be used as an alternative method. Although this draft OECD guideline may appropriate be suitable to determine Pow for those ionisable substances, in some cases it is more appropriate to use the HPLC method at an environmentally relevant pH (see paragraph 9).

PRINCIPLE OF THE METHOD

• 6.Reverse phase HPLC is performed on analytical columns packed with a commercially available solid phase containing long hydrocarbon chains (e.g. C8, C18) chemically bound onto silica.

• 7.A chemical injected on such a column partitions between the mobile solvent phase and the hydrocarbon stationary phase as it is transported along the column by the mobile phase. The substances are retained in proportion to their hydrocarbon-water partition coefficient, with hydrophilic substances eluted first and lipophilic substances last. The retention time is described by the capacity factor k given by the expression:

Image omitted; see official source

• where tR is the retention time of the test substance, and t0 is the dead-time, i.e. the average time a solvent molecule needs to pass the column. Quantitative analytical methods are not required and only the determination of retention times is necessary.

• 8.The octanol/water partition coefficient of a test substance can be computed by experimentally determining its capacity factor k and then inputting k into the following equation:

Image omitted; see official source

• where

a, b	=	linear regression coefficients.

• The equation above can be obtained by linearly regressing the log of octanol/water partition coefficients of reference substances against the log of capacity factors of the reference substances.

• 9.Reverse phase HPLC method enables partition coefficients to be estimated in the log Pow range between 0 and 6, but can be expanded to cover the log Pow range between 6 and 10 in exceptional cases. This may require that the mobile phase is modified (3). The method is not applicable to strong acids and bases, metal complexes, substances which react with the eluent, or surface-active agents. Measurements can be performed on ionisable substances in their non-ionised form (free acid or free base) only by using an appropriate buffer with a pH below the pKa for a free acid or above the pKa for a free base. Alternatively, the pH-metric method for the testing of ionisable substances (6) may become available and could be used as an alternative method (6). If the log Pow value is determined for the use in environmental hazard classification or in environmental risk assessment, the test should be performed in the pH range relevant for the natural environment, i.e. in the pH range of 5,0 - 9.

• 10.In some cases impurities can make the interpretation of the results difficult due to uncertainty in peak assignments. For mixtures which result in an unresolved band, upper and lower limits of log Pow, and the area % of each log Pow peak should be reported. For mixtures which are a group of homologues, the weighted average log Pow should also be stated (7), calculated based on the single Pow values and the corresponding area % values (8). All peaks that contribute an area of 5 % or more to the total peak area should be taken into consideration in the calculation (9):

Image omitted; see official source

• The weighed average log Pow is valid only for substances or mixtures (e.g. tall oils) consisting of homologues (e.g. series of alkanes). Mixtures can be measured with meaningful results, provided that the analytical detector used has the same sensitivity towards all the substances in the mixture and that they can be adequately resolved.

INFORMATION ON THE TEST SUBSTANCE

• 11.The dissociation constant, structural formula, and solubility in the mobile phase should be known before the method is used. In addition, information on hydrolysis would be helpful.

QUALITY CRITERIA

• 12.In order to increase the confidence in the measurement, duplicate determinations must be made.

• — Repeatability: The value of log Pow derived from repeated measurements made under identical conditions and using the same set of reference substances should fall within a range of ± 0,1 log units.

• — Reproducibility: If the measurements are repeated with a different set of reference substances, results may differ. Typically, the correlation coefficient R for the relationship between log k and log Pow for a set of test substances is around 0,9, corresponding to an octanol/water partition coefficient of log Pow ± 0,5 log units.

• 13.The inter-laboratory comparison test has shown that with the HPLC method log Pow values can be obtained to within ± 0,5 units of the Shake-Flask values (2). Other comparisons can be found in the literature (4)(5)(10)(11)(12). Correlation graphs based on structurally related reference substances give the most accurate results (13).

REFERENCE SUBSTANCES

• 14.In order to correlate the measured capacity factor k of a substance with its Pow, a calibration graph using at least 6 points has to be established (see paragraph 24). It is up to the user to select the appropriate reference substances. The reference substances should normally have log Pow values which encompass the log Pow of the test substance, i.e. at least one reference substance should have a Pow above that of the test substance, and another a Pow below that of the test substance. Extrapolation should only be used in exceptional cases. It is preferable that these reference substances should be structurally related to the test substance. log Pow values of the reference substances used for the calibration should be based on reliable experimental data. However, for substances with high log Pow (normally more than 4), calculated values may be used unless reliable experimental data are available. If extrapolated values are used a limit value should be quoted.

• 15.Extensive lists of log Pow values for many groups of chemicals are available (14)(15). If data on the partition coefficients of structurally related substances are not available, a more general calibration, established with other reference substances, may be used. Recommended reference substances and their Pow values are listed in Table 1. For ionisable substances the values given apply to the non-ionised form. The values were checked for plausibility and quality during the inter-laboratory comparison test.

Table 1

Recommended reference substances

	CAS Number	Reference substance	log Pow	pKa
1	78-93-3	2-Butanone (Methylethylketone)	0,3
2	1122-54-9	4-Acetylpyridine	0,5
3	62-53-3	Aniline	0,9
4	103-84-4	Acetanilide	1,0
5	100-51-6	Benzyl alcohol	1,1
6	150-76-5	4-Methoxyphenol	1,3	pKa = 10,26
7	122-59-8	Phenoxyacetic acid	1,4	pKa = 3,12
8	108-95-2	Phenol	1,5	pKa = 9,92
9	51-28-5	2,4-Dinitrophenol	1,5	pKa = 3,96
10	100-47-0	Benzonitrile	1,6
11	140-29-4	Phenylacetonitrile	1,6
12	589-18-4	4-Methylbenzyl alcohol	1,6
13	98-86-2	Acetophenone	1,7
14	88-75-5	2-Nitrophenol	1,8	pKa = 7,17
15	121-92-6	3-Nitrobenzoic acid	1,8	pKa = 3,47
16	106-47-8	4-Chloroaniline	1,8	pKa = 4,15
17	98-95-3	Nitrobenzene	1,9
18	104-54-1	Cinnamyl alcohol (Cinnamic alcohol)	1,9
19	65-85-0	Benzoic acid	1,9	pKa = 4,19
20	106-44-5	p-Cresol	1,9	pKa = 10,17
21	140-10-3 (trans)	Cinnamic acid	2,1	pKa = 3,89 (cis) 4,44 (trans)
22	100-66-3	Anisole	2,1
23	93-58-3	Methyl benzoate	2,1
24	71-43-2	Benzene	2,1
25	99-04-7	3-Methylbenzoic acid	2,4	pKa = 4,27
26	106-48-9	4-Chlorophenol	2,4	pKa = 9,1
27	79-01-6	Trichloroethylene	2,4
28	1912-24-9	Atrazine	2,6
29	93-89-0	Ethyl benzoate	2,6
30	1194-65-6	2,6-Dichlorobenzonitrile	2,6
31	535-80-8	3-Chlorobenzoic acid	2,7	pKa = 3,82
32	108-88-3	Toluene	2,7
33	90-15-3	1-Naphthol	2,7	pKa = 9,34
34	608-27-5	2,3-Dichloroaniline	2,8
35	108-90-7	Chlorobenzene	2,8
36	1746-13-0	Allyl phenyl ether	2,9
37	108-86-1	Bromobenzene	3,0
38	100-41-4	Ethylbenzene	3,2
39	119-61-9	Benzophenone	3,2
40	92-69-3	4-Phenylphenol	3,2	pKa = 9,54
41	89-83-8	Thymol	3,3
42	106-46-7	1,4-Dichlorobenzene	3,4
43	122-39-4	Diphenylamine	3,4	pKa = 0,79
44	91-20-3	Naphthalene	3,6
45	93-99-2	Phenyl benzoate	3,6
46	98-82-8	Isopropylbenzene	3,7
47	88-06-2	2,4,6-Trichlorophenol	3,7	pKa = 6
48	92-52-4	Biphenyl	4,0
49	120-51-4	Benzyl benzoate	4,0
50	88-85-7	2,4-Dinitro-6-sec-butylphenol	4,1
51	120-82-1	1,2,4-Trichlorobenzene	4,2
52	143-07-7	Dodecanoic acid	4,2	pKa = 5,3
53	101-84-8	Diphenyl ether	4,2
54	85-01-8	Phenanthrene	4,5
55	104-51-8	n-Butylbenzene	4,6
56	103-29-7	Dibenzyl	4,8
57	3558-69-8	2,6-Diphenylpyridine	4,9
58	206-44-0	Fluoranthene	5,1
59	603-34-9	Triphenylamine	5,7
60	50-29-3	DDT	6,5

DESCRIPTION OF THE METHOD

Preliminary estimate of the partition coefficient

• 16.If it is necessary, the partition coefficient of the test substance may be estimated preferably by using a calculation method (see Appendix, or where appropriate, by using the ratio of the solubility of the test substance in the pure solvents.

Apparatus

• 17.A liquid-phase chromatograph fitted with a low-pulse pump and a suitable detection system is required. A UV detector, using a wavelength of 210 nm, or an RI detector is applicable to the wide variety of chemical groups. The presence of polar groups in the stationary phase may seriously impair the performance of the HPLC column. Therefore, stationary phases should have a minimal percentage of polar groups (16). Commercial microparticulate reverse-phase packing or ready-packed columns can be used. A guard column may be positioned between the injection system and the analytical column.

Mobile phase

• 18.HPLC-grade methanol and distilled or de-ionised water are used to prepare the eluting solvent, which is degassed before use. Isocratic elution should be employed. Methanol/water ratios with minimum water content of 25 % should be used. Typically a 3:1 (v/v) methanol-water mixture is satisfactory for eluting substances with a log P of 6 within an hour, at a flow rate of 1 ml/min. For substances with a log P above 6 it may be necessary to shorten the elution time (and those of the reference substances) by decreasing the polarity of the mobile phase or the column length.

• 19.The test substance and the reference substances must be soluble in the mobile phase in sufficient concentration to allow their detection. Additives may be used with the methanol-water mixture in exceptional cases only, since they will change the properties of the column. In these cases it must be confirmed that the retention time of the test and reference substances are not influenced. If methanol-water is not appropriate, other organic solvent-water mixtures can be used, e.g. ethanol-water, acetonitrile-water or isopropyl alcohol (2-propanol)-water.

• 20.The pH of the eluent is critical for ionisable substances. It should be within the operating pH range of the column, usually between 2 and 8. Buffering is recommended. Care must be taken to avoid salt precipitation and column deterioration which occur with some organic phase/buffer mixtures. HPLC measurements with silica-based stationary phases above pH 8 are not normally advisable since the use of an alkaline mobile phase may cause rapid deterioration in the performance of the column.

Solutes

• 21.The test and reference substances must be sufficiently pure in order to assign the peaks in the chromatograms to the respective substances. Substances to be used for test or calibration purposes are dissolved in the mobile phase if possible. If a solvent other than the mobile phase is used to dissolve the test and reference substances, the mobile phase should be used for the final dilution prior to injection.

Test conditions

• 22.The temperature during the measurement should not vary by more than ± 1 °C.

Determination of dead time to

• 23.The dead time t0 can be measured by using unretained organic substances (e.g. thiourea or formamide). A more precise dead time can be derived from the retention times measured or a set of approximately seven members of a homologous series (e.g. n-alkyl methyl ketones) (17). The retention times tR (nC + 1) are plotted against tR (nC), where nC is the number of carbon atoms. A straight line, tR (nC + 1) = A tR (nC) + (1 – A)t0, is obtained, where A, representing k(nC + 1)/k(nC), is constant. The dead time t0 is obtained from the intercept (1 – A)t0 and the slope A.

Regression Equation

• 24.The next step is to plot a correlation log k versus log P for appropriate reference substances with log P values near the value expected for the test substance. In practice, from 6 to 10 reference substances are injected simultaneously. The retention times are determined, preferably on a recording integrator linked to the detection system. The corresponding logarithms of the capacity factors, log k, are plotted as a function of log P. The regression equation is performed at regular intervals, at least once daily, so that account can be taken of possible changes in column performance.

DETERMINATION OF THE POW OF THE TEST SUBSTANCE

• 25.The test substance is injected in the smallest detectable quantities. The retention time is determined in duplicate. The partition coefficient of the test substance is obtained by interpolation of the calculated capacity factor on the calibration graph. For very low and very high partition coefficients extrapolation is necessary. Especially in these cases attention must be given to the confidence limits of the regression line. If the retention time of sample is outside the range of retention times obtained for the standards, a limit value should be quoted.

DATA AND REPORTING

Test report

• 26.The following must be included in the report:

• — if determined the preliminary estimate of the partition coefficient, the estimated values and the method used; and if a calculation method was used, its full description including identification of the data base and detailed information on the choice of fragments;

• — test and reference substances: purity, structural formula and CAS number,

• — description of equipment and operating conditions: analytical column, guard column,

• — mobile phase, means of detection, temperature range, pH;

• — elution profiles (chromatograms);

• — deadtime and how it was measured;

• — retention data and literature log Pow values for reference substances used in calibration;

• — details on fitted regression line (log k versus log Pow) and the correlation coefficient of the line including confidence intervals;

• — average retention data and interpolated log Pow value for the test substance;

• — in case of a mixture: elution profile chromatogram with indicated cut-offs;

• — log Pow values relative to area % of the log Pow peak;

• — calculation using a regression line;

• — calculated weighted average log Pow values, when appropriate.

LITERATURE

• (1)C.V. Eadsforth and P. Moser. (1983). Assessment of Reverse Phase Chromatographic Methods for Determining Partition Coefficients. Chemosphere. 12, 1459.

• (2)W. Klein, W. Kördel, M. Weiss and H.J. Poremski. (1988). Updating of the OECD Test Guideline 107 Partition Coefficient n-Octanol-Water, OECD Laboratory Intercomparison Test on the HPLC Method. Chemosphere. 17, 361.

• (3)C.V. Eadsforth. (1986). Application of Reverse H.P.L.C. for the Determination of Partition Coefficient. Pesticide Science. 17, 311.

• (4)H. Ellgehausen, C. D'Hondt and R. Fuerer (1981). Reversed-phase chromatography as a general method for determining octan-1-ol/water partition coefficients. Pesticide. Science. 12, 219.

• (5)B. McDuffie (1981). Estimation of Octanol Water Partition Coefficients for Organic Pollutants Using Reverse Phase High Pressure Liquid Chromatography. Chemosphere. 10, 73.

• (6)OECD (2000). Guideline for Testing of Chemicals — Partition Coefficient (n-octanol/water): pH-metric Method for Ionisable Substances. Draft Guideline, November 2000.

• (7)OSPAR (1995). ‘Harmonised Offshore Chemicals Notification Format (HOCFN) 1995’, Oslo and Paris Conventions for the Prevention of Marine Pollution Programmes and Measures Committee (PRAM), Annex 10, Oviedo, 20–24 February 1995.

• (8)M. Thatcher, M. Robinson, L. R. Henriquez and C. C. Karman. (1999). An User Guide for the Evaluation of Chemicals Used and Discharged Offshore, A CIN Revised CHARM III Report 1999. Version 1.0, 3. August.

• (9)E. A. Vik, S. Bakke and K. Bansal. (1998). Partitioning of Chemicals. Important Factors in Exposure Assessment of Offshore Discharges. Environmental Modelling & Software Vol. 13, pp. 529-537.

• (10)L.O. Renberg, S.G. Sundstroem and K. Sundh-Nygård. (1980). Partition coefficients of organic chemicals derived from reversed-phase thin-layer chromatography. Evaluation of methods and application on phosphate esters, polychlorinated paraffins and some PCB-substitutes. Chemosphere. 9, 683.

• (11)W.E. Hammers, G.J.Meurs and C.L. De-Ligny. (1982). Correlations between liquid chromatographic capacity ratio data on Lichrosorb RP-18 and partition coefficients in the octanol-water system. J. Chromatography 247, 1.

• (12)J.E. Haky and A.M. Young. (1984). Evaluation of a simple HPLC correlation method for the estimation of the octanol-water partition coefficients of organic compounds. J. Liq. Chromatography. 7, 675.

• (13)S. Fujisawa and E. Masuhara. (1981). Determination of Partition Coefficients of Acrylates Methacrylates and Vinyl Monomers Using High Performance Liquid Chromatography. Journal of Biomedical Materials Research. 15, 787.

• (14)C. Hansch and A. J. Leo. (1979). Substituent Constants for Correlation Analysis in Chemistry and Biology. John Willey, New York.

• (15)C. Hansch, chairman; A.J. Leo, dir. (1982). Log P and Parameter Database: A tool for the quantitative prediction of bioactivity — Available from Pomona College Medical Chemistry Project, Pomona College, Claremont, California 91711.

• (16)R. F. Rekker, H. M. de Kort. (1979). The hydrophobic fragmental constant: An extension to a 1 000 data point set. Eur. J. Med. Chem. — Chim. Ther. 14, 479.

• (17)G.E. Berendsen, P.J. Schoenmakers, L. de Galan, G. Vigh, Z. Varga-Puchony, and J. Inczédy. (1980). On determination of hold-up time in reversed-phase liquid chromatography. J. Liq. Chromato. 3, 1669.

Appendix

POW calculation methods

INTRODUCTION

• 1.This appendix provides a short introduction to the calculation of Pow. For further information the reader is referred to textbooks (1)(2).

• 2.Calculated values of Pow are used for:

• — deciding which experimental method to use: Shake Flask method for log Pow between – 2 and 4 and HPLC method for log Pow between 0 and 6;

• — selecting conditions to be used in HPLC (reference substances, methanol/water ratio);

• — checking the plausibility of values obtained through experimental methods;

• — providing an estimate when experimental methods cannot be applied.

Principle of calculation methods

• 3.The calculation methods suggested here are based on the theoretical fragmentation of the molecule into suitable substructures for which reliable log Pow increments are known. The log Pow is obtained by summing the fragment values and the correction terms for intramolecular interactions. Lists of fragment constants and correction terms are available (1)(2)(3)(4)(5)(6). Some are regularly updated (3).

Reliability of calculated values

• 4.In general, the reliability of calculation methods decreases as the complexity of the substance under study increases. In the case of simple molecules of low molecular weight and with one or two functional groups, a deviation of 0,1 to 0,3 log Pow units between the results of the different fragmentation methods and the measured values can be expected. The margin of error will depend on the reliability of the fragment constants used, the ability to recognise intramolecular interactions (e.g. hydrogen bonds) and the correct use of correction terms. In the case of ionising substances the charge and degree of ionisation must be taken into consideration (10).

Fujita-Hansch π-method

• 5.The hydrophobic substituent constant, π, originally introduced by Fujita et al. (7) is defined as:

• ^πX = log Pow (PhX) – log Pow (PhH)

• where PhX is an aromatic derivative and PhH the parent substance.

e.g.	πCl	= log Pow (C6H5Cl) – log Pow (C6H6) = 2,84 – 2,13 = 0,71

• The π-method is primarily of interest for aromatic substances. π-values for a large number of substituents are available (4)(5).

Rekker method

• 6.Using the Rekker method (8) the log Pow value is calculated as:

Image omitted; see official source

• where ai is the number of times a given fragment occurs in the molecule and fi is the log Pow increment of the fragment. The interaction terms can be expressed as an integral multiple of one single constant Cm (so-called ‘magic constant’). The fragment constants fi and Cm have been determined from a list of 1 054 experimental Pow values of 825 substances using multiple regression analysis (6)(8). The determination of the interaction terms is carried out according to set rules (6)(8)(9).

Hansch-Leo method

• 7.Using the Hansch and Leo method (4), the log Pow value is calculated as:

Image omitted; see official source

• where fi is a fragment constant, Fj a correction term (factor), ai and bj the corresponding frequency of occurence. Lists of atomic and group fragmental values and of correction terms Fj were derived by trial and error from experimental Pow values. The correction terms have been divided into several different classes (1)(4). Sofware packages have been developed to take into account all the rules and correction terms (3).

COMBINED METHOD

• 8.The calculation of log Pow of complex molecules can be considerably improved, if the molecule is dissected into larger substructures for which reliable log Pow values are available, either from tables (3)(4) or by existing measurements. Such fragments (e.g. heterocycles, anthraquinone, azobenzene) can then be combined with the Hansch- π values or with Rekker or Leo fragment constants.

Remarks:

• (i) The calculation methods are only applicable to partly or fully ionised substances when the necessary correction factors are taken into account.

• (ii) If the existence of intramolecular hydrogen bonds can be assumed, the corresponding correction terms (approx. + 0,6 to + 1,0 log Pow units) must be added (1). Indications on the presence of such bonds can be obtained from stereo models or spectroscopic data.

• (iii) If several tautomeric forms are possible, the most likely form should be used as the basis of the calculation.

• (iv) The revisions of lists of fragment constants should be followed carefully.

LITERATURE ON CALCULATION METHODS

• (1)W.J. Lyman, W.F. Reehl and D.H. Rosenblatt (ed.). Handbook of Chemical Property Estimation Methods, McGraw-Hill, New York (1982).

• (2)W.J. Dunn, J.H. Block and R.S. Pearlman (ed.). Partition Coefficient, Determination and Estimation, Pergamon Press, Elmsford (New York) and Oxford (1986).

• (3)Pomona College, Medicinal Chemistry Project, Claremont, California 91711, USA, Log P Database and Med. Chem. Software (Program CLOGP-3).

• (4)C. Hansch and A.J. Leo. Substituent Constants for Correlation Analysis in Chemistry and Biology, John Wiley, New York (1979).

• (5)Leo, C. Hansch and D. Elkins. (1971) Partition coefficients and their uses. Chemical. Reviews. 71, 525.

• (6)R. F. Rekker, H. M. de Kort. (1979). The hydrophobic fragmental constant: An extension to a 1 000 data point set. Eur. J. Med. Chem. — Chim. Ther. 14, 479.

• (7)Toshio Fujita, Junkichi Iwasa & Corwin Hansch (1964). A New Substituent Constant, π, Derived from Partition Coefficients. J. Amer. Chem. Soc. 86, 5175.

• (8)R.F. Rekker. The Hydrophobic Fragmental Constant, Pharmacochemistry Library, Vol. 1, Elsevier, New York (1977).

• (9)C.V. Eadsforth and P. Moser. (1983). Assessment of Reverse Phase Chromatographic Methods for Determining Partition Coefficients. Chemosphere. 12, 1459.

• (10)R.A. Scherrer. ACS — Symposium Series 255, p. 225, American Chemical Society, Washington, D.C. (1984).

A.25.   DISSOCIATION CONSTANTS IN WATER (TITRATION METHOD — SPECTROPHOTOMETRIC METHOD — CONDUCTOMETRIC METHOD)

INTRODUCTION

This test method is equivalent to OECD test guideline 112 (1981)

Prerequisites

• — Suitable analytical method

• — Water solubility

Guidance information

• — Structural formula

• — Electrical conductivity for conductometric method

Qualifying statements

• — All test methods may be carried out on pure or commercial grade substances. The possible effects of impurities on results should be considered.

• — The titration method is not suitable for low solubility substances (see Test solutions, below).

• — The spectrophotometric method is only applicable to substances having appreciably different UV/VIS-absorption spectra for the dissociated and undissociated forms. This method may also be suitable for low solubility substances and for non-acid/base dissociations, e.g. complex formation.

• — In cases where the Onsager equation holds, the conductometric method may be used, even at moderately low concentrations and even in cases for non-acid/base equilibria.

Standard documents

This test method is based on methods given in the references listed in the section ‘Literature’ and on the Preliminary Draft Guidance for Premanufacture Notification EPA, August 18, 1978.

METHOD — INTRODUCTION, PURPOSE, SCOPE, RELEVANCE, APPLICATION AND LIMITS OF TEST

The dissociation of a substance in water is of importance in assessing its impact upon the environment. It governs the form of the substance which in turn determines its behaviour and transport. It may affect the adsorption of the chemical on soils and sediments and absorption into biological cells.

Definitions and units

Dissociation is the reversible splitting into two or more chemical species which may be ionic. The process is indicated generally by

RX⇌R ⁺+ X ^–

and the concentration equilibrium constant governing the reaction is

Image omitted; see official source

For example, in the particular case where R is hydrogen (the substance is an acid), the constant is

Image omitted; see official source

or

Image omitted; see official source

Reference substances

The following reference substances need not be employed in all cases when investigating a new substance. They are provided primarily so that calibration of the method may be performed from time to time and to offer the chance to compare the results when another method is applied.

	pKa [^E0011]	Temp. in °C
p-Nitrophenol	7,15	25 [^E0011]
Benzoic acid	4,12	20
p-Chloroaniline	3,93	20

No value for 20 °C is available, but it can be assumed that the variability of measurement results is higher than the temperature dependence to be expected.

It would be useful to have a substance with several pKs as indicated in Principle of the method, below. Such a substance could be:

Citric acid	pKa (8)	Temp. in °C
	(1) 3,14	20
	(2) 4,77	20
	(3) 6,39	20

Principle of the test method

The chemical process described is generally only slightly temperature dependent in the environmentally relevant temperature range. The determination of the dissociation constant requires a measure of the concentrations of the dissociated and undissociated forms of the chemical substance. From the knowledge of the stoichiometry of the dissociation reaction indicated in Definitions and units, above, the appropriate constant can be determined. In the particular case described in this test method the substance is behaving as an acid or a base, and the determination is most conveniently done by determining the relative concentrations of the ionised and unionised forms of the substance and the pH of the solution. The relationship between these terms is given in the equation for pKa in Definitions and units, above. Some substances exhibit more than one dissociation constant and similar equations can be developed. Some of the methods described herein are also suitable for non-acid/base dissociation.

Quality criteria

Repeatability

The dissociation constant should be replicated (a minimum of three determinations) to within ± 0,1 log units.

DESCRIPTION OF THE TEST PROCEDURES

There are two basic approaches to the determination of pKa. One involves titrating a known amount of substance with standard acid or base, as appropriate; the other involves determining the relative concentration of the ionised and unionised forms and its pH dependence.

Preparations

Methods based on those principles may be classified as titration, spectrophotometric and conductometric procedures.

Test solutions

For the titration method and conductometric method the chemical substance should be dissolved in distilled water. For spectrophotometric and other methods buffer solutions are used. The concentration of the test substance should not exceed the lesser of 0,01 M or half the saturation concentration, and the purest available form of the substance should be employed in making up the solutions. If the substance is only sparingly soluble, it may be dissolved in a small amount of a water-miscible solvent prior to adding to the concentrations indicated above.

Solutions should be checked for the presence of emulsions using a Tyndall beam, especially if a co-solvent has been used to enhance solubility. Where buffer solutions are used, the buffer concentration should not exceed 0,05 M.

Test conditions

Temperature

The temperature should be controlled to at least ± 1 °C. The determination should preferably be carried out at 20 °C.

If a significant temperature dependence is suspected, the determination should be carried out at least at two other temperatures. The temperature intervals should be 10 °C in this case and the temperature control ± 0,1 °C.

Analyses

The method will be determined by the nature of the substance being tested. It must be sufficiently sensitive to allow the determination of the different species at each test solution concentration.

Performance of the test

Titration method

The test solution is determined by titration with the standard base or acid solution as appropriate, measuring the pH after each addition of titrant. At least 10 incremental additions should be made before the equivalence point. If equilibrium is reached sufficiently rapidly, a recording potentiometer may be used. For this method both the total quantity of substance and its concentration need to be accurately known. Precautions must be taken to exclude carbon dioxide. Details of procedure, precautions, and calculation are given in standard tests, e.g. references (1), (2), (3), (4).

Spectrophotometric method

A wavelength is found where the ionised and unionised forms of the substance have appreciably different extinction coefficients. The UV/VIS absorption spectrum is obtained from solutions of constant concentration under a pH condition where the substance is essentially unionised and fully ionised and at several intermediate pHs. This may be done, either by adding increments of concentrated acid (base) to a relatively large volume of a solution of the substance in a multicomponent buffer, initially at high (low) pH (ref. 5), or by adding equal volumes of a stock solution of the substance in e.g. water, methanol, to constant volumes of various buffer solutions covering the desired pH range. From the pH and absorbance values at the chosen wavelength, a sufficient number of values for the pKa is calculated using data from at least 5 pHs where the substance is at least 10 per cent and less than 90 per cent ionised. Further experimental details and method of calculation are given in reference (1).

Conductometric method

Using a cell of small, known cell constant, the conductivity of an approximately 0,1 M solution of the substance in conductivity water is measured. The conductivities of a number of accurately-made dilutions of this solution are also measured. The concentration is halved each time, and the series should cover at least an order of magnitude in concentration. The limiting conductivity at infinite dilution is found by carrying out a similar experiment with the Na salt and extrapolating. The degree of dissociation may then be calculated from the conductivity of each solution using the Onsager equation, and hence using the Ostwald Dilution Law the dissociation constant may be calculated as K = α²C/(1 – α) where C is the concentration in moles per litre and α is the fraction dissociated. Precautions must be taken to exclude CO2. Further experimental details and method of calculation are given in standard texts and references (1), (6) and (7).

DATA AND REPORTING

Treatment of results

Titration method

The pKa is calculated for 10 measured points on the titration curve. The mean and standard deviation of such pKa values are calculated. A plot of pH versus volume of standard base or acid should be included along with a tabular presentation.

Spectrophotometric methods

The absorbance and pH are tabulated from each spectrum. At least five values for the pKa are calculated from the intermediate spectra data points, and the mean and standard deviation of these results are also calculated.

Conductometric method

The equivalent conductivity Λ is calculated for each acid concentration and for each concentration of a mixture of one equivalent of acid, plus 0,98 equivalent of carbonate-free sodium hydroxide. The acid is in excess to prevent an excess of OH^– due to hydrolysis. 1/Λ is plotted against √C and Λo of the salt can be found by extrapolation to zero concentration.

Λo of the acid can be calculated using literature values for H⁺ and Na⁺. The pKa can be calculated from α = Λi/Λo and Ka = α²C/(1 – α) for each concentration. Better values for Ka can be obtained by making corrections for mobility and activity. The mean and standard deviations of the pKa values should be calculated.

Test report

All raw data and calculated pKa values should be submitted together with the method of calculation (preferably in a tabulated format, such as suggested in ref. 1) as should the statistical parameters described above. For titration methods, details of the standardisation of titrants should be given.

For the spectrophotometric method, all spectra should be submitted. For the conductometric method, details of the cell constant determination should be reported. Information on technique used, analytical methods and the nature of any buffers used should be given.

The test temperature(s) should be reported.

LITERATURE:

• (1) Albert, A. & Sergeant, E.P.: Ionization Constants of Acids and Bases, Wiley, Inc., New York, 1962.

• (2) Nelson, N.H. & Faust, S.D.: Acidic dissociation constants of selected aquatic herbicides, Env. Sci. Tech. 3, II, pp. 1186-1188 (1969).

• (3) ASTM D 1293 — Annual ASTM Standards, Philadelphia, 1974.

• (4) Standard Method 242. APHA/AWWA/WPCF, Standard Methods for the Examination of Water and Waste Water, 14th Edition, American Public Health Association, Washington, D.C., 1976.

• (5) Clark, J. & Cunliffe, A.E.: Rapid spectrophotometric measurement of ionisation constants in aqueous solution. Chem. Ind. (London) 281, (March 1973).

• (6) ASTM D 1125 — Annual ASTM Standards, Philadelphia, 1974.

• (7) Standard Method 205 — APHA/AWWA/NPCF (see above (4)).

• (8) Handbook of Chemistry and Physics, 60th ed. CRC-Press, Boca Raton, Florida, 33431 (1980).

PART B: METHODS FOR THE DETERMINATION OF TOXICITY AND OTHER HEALTH EFFECTS

TABLE OF CONTENTS	TABLE OF CONTENTS
	GENERAL INTRODUCTION
B.1 bis.	ACUTE ORAL TOXICITY — FIXED DOSE PROCEDURE
B.1 tris.	ACUTE ORAL TOXICITY — ACUTE TOXIC CLASS METHOD
B.2.	ACUTE INHALATION TOXICITY
B.3.	ACUTE TOXICITY (DERMAL)
B.4.	ACUTE DERMAL IRRITATION/CORROSION
B.5.	ACUTE EYE IRRITATION/CORROSION
B.6.	SKIN SENSITISATION
B.7.	REPEATED DOSE 28-DAY ORAL TOXICITY STUDY IN RODENTS
B.8.	SUBACUTE INHALATION TOXICITY: 28-DAY STUDY
B.9.	REPEATED DOSE (28 DAYS) TOXICITY (DERMAL)
B.10.	IN VITRO MAMMALIAN CHROMOSOMAL ABERRATION TEST
B.11.	MAMMALIAN BONE MARROW CHROMOSOMAL ABERRATION TEST
B.12.	MAMMALIAN ERYTHROCYTE MICRONUCLEUS TEST
B.13/14.	MUTAGENICITY: REVERSE MUTATION TEST USING BACTERIA
B.17.	IN VITRO MAMMALIAN CELL GENE MUTATION TESTS USING THE HPRT AND XPRT GENES
B.21.	IN VITRO MAMMALIAN CELL TRANSFORMATION TESTS
B.22.	RODENT DOMINANT LETHAL TEST
B.23.	MAMMALIAN SPERMATOGONIAL CHROMOSOMAL ABERRATION TEST
B.25.	MOUSE HERITABLE TRANSLOCATION
B.26.	SUB-CHRONIC ORAL TOXICITY TEST REPEATED DOSE 90 — DAY ORAL TOXICITY STUDY IN RODENTS
B.27.	SUB-CHRONIC ORAL TOXICITY TEST REPEATED DOSE 90 — DAY ORAL TOXICITY STUDY IN NON-RODENTS
B.28.	SUB-CHRONIC DERMAL TOXICITY STUDY 90-DAY REPEATED DERMAL DOSE STUDY USING RODENT SPECIES
B.29.	SUBCHRONIC INHALATION TOXICITY: 90-DAY STUDY
B.30.	CHRONIC TOXICITY STUDIES
B.31.	PRENATAL DEVELOPMENTAL TOXICITY STUDY
B.32.	CARCINOGENICITY STUDIES
B.33.	COMBINED CHRONIC TOXICITY/CARCINOGENICITY STUDIES
B.34.	ONE-GENERATION REPRODUCTION TOXICITY TEST
B.35.	TWO-GENERATION REPRODUCTION TOXICITY STUDY
B.36.	TOXICOKINETICS
B.37.	DELAYED NEUROTOXICITY OF ORGANOPHOSPHORUS SUBSTANCES FOLLOWING ACUTE EXPOSURE
B.38.	DELAYED NEUROTOXICITY OF ORGANOPHOSPHORUS SUBSTANCES 28 DAY REPEATED DOSE STUDY
B.39.	UNSCHEDULED DNA SYNTHESIS (UDS) TEST WITH MAMMALIAN LIVER CELLS IN VIVO
B.40.	IN VITRO SKIN CORROSION: TRANSCUTANEOUS ELECTRICAL RESISTANCE TEST METHOD (TER)
B.40 BIS.	IN VITRO SKIN CORROSION: RECONSTRUCTED HUMAN EPIDERMIS (RhE) TEST METHOD
B.41.	IN VITRO 3T3 NRU PHOTOTOXICITY TEST
B.42.	SKIN SENSITISATION: LOCAL LYMPH NODE ASSAY
B.43.	NEUROTOXICITY STUDY IN RODENTS
B.44.	SKIN ABSORPTION: IN VIVO METHOD
B.45.	SKIN ABSORPTION: IN VITRO METHOD
B.46.	IN VITRO SKIN IRRITATION: RECONSTRUCTED HUMAN EPIDERMIS TEST METHOD
B.47.	BOVINE CORNEAL OPACITY AND PERMEABILITY TEST METHOD FOR IDENTIFYING (I) CHEMICALS INDUCING SERIOUS EYE DAMAGE AND (II) CHEMICALS NOT REQUIRING CLASSIFICATION FOR EYE IRRITATION OR SERIOUS EYE DAMAGE
B.48.	ISOLATED CHICKEN EYE TEST METHOD FOR IDENTIFYING I) CHEMICALS INDUCING SERIOUS EYE DAMAGE AND II) CHEMICALS NOT REQUIRING CLASSIFICATION FOR EYE IRRITATION OR SERIOUS EYE DAMAGE
B.49.	IN VITRO MAMMALIAN CELL MICRONUCLEUS TEST
B.50.	SKIN SENSITISATION: LOCAL LYMPH NODE ASSAY: DA
B.51.	SKIN SENSITISATION: LOCAL LYMPH NODE ASSAY: BrdU-ELISA
B.52.	ACUTE INHALATION TOXICITY — ACUTE TOXIC CLASS METHOD
B.53.	DEVELOPMENTAL NEUROTOXICITY STUDY
B.54.	UTEROTROPHIC BIOASSAY IN RODENTS: A SHORT-TERM SCREENING TEST FOR OESTROGENIC PROPERTIES
B.55.	HERSHBERGER BIOASSAY IN RATS: A SHORT-TERM SCREENING ASSAY FOR (ANTI)ANDROGENIC PROPERTIES
B.56.	EXTENDED ONE-GENERATION REPRODUCTIVE TOXICITY STUDY
B.57.	H295R STEROIDOGENESIS ASSAY
B.58.	TRANSGENIC RODENT SOMATIC AND GERM CELL GENE MUTATION ASSAYS
B.59.	IN CHEMICO SKIN SENSITISATION: DIRECT PEPTIDE REACTIVITY ASSAY (DPRA)
B.60.	IN VITRO SKIN SENSITISATION: ARE-NRF2 LUCIFERASE TEST METHOD
B.61.	FLUORESCEIN LEAKAGE TEST METHOD FOR IDENTIFYING OCULAR CORROSIVES AND SEVERE IRRITANTS
B.62.	IN VIVO MAMMALIAN ALKALINE COMET ASSAY
B.63.	REPRODUCTION/DEVELOPMENTAL TOXICITY SCREENING TEST
B.64.	COMBINED REPEATED DOSE TOXICITY STUDY WITH THE REPRODUCTION/DEVELOPMENTAL TOXICITY SCREENING TEST
B.65.	IN VITRO MEMBRANE BARRIER TEST METHOD FOR SKIN CORROSION
B.66.	STABLY TRANSFECTED TRANSACTIVATION IN VITRO ASSAYS TO DETECT ESTROGEN RECEPTOR AGONISTS AND ANTAGONISTS
B.67.	IN VITRO MAMMALIAN CELL GENE MUTATION TESTS USING THE THYMIDINE KINASE GENE
B.68.	SHORT TIME EXPOSURE IN VITRO TEST METHOD FOR IDENTIFYING i) CHEMICALS INDUCING SERIOUS EYE DAMAGE AND ii) CHEMICALS NOT REQUIRING CLASSIFICATION FOR EYE IRRITATION OR SERIOUS EYE DAMAGE
B.69.	RECONSTRUCTED HUMAN CORNEA-LIKE EPITHELIUM (RhCE) TEST METHOD FOR IDENTIFYING CHEMICALS NOT REQUIRING CLASSIFICATION AND LABELLING FOR EYE IRRITATION OR SERIOUS EYE DAMAGE
B.70.	HUMAN RECOMBINANT ESTROGEN RECEPTOR (hrER) IN VITRO ASSAYS TO DETECT CHEMICALS WITH ER BINDING AFFINITY
B.71.	IN VITRO SKIN SENSITISATION ASSAYS ADDRESSING THE KEY EVENT ON ACTIVATION OF DENDRITIC CELLS ON THE ADVERSE OUTCOME PATHWAY (AOP) FOR SKIN SENSITISATION

GENERAL INTRODUCTION

A. CHARACTERISATION OF THE TEST SUBSTANCE

The composition of the test substance, including major impurities, and its relevant physico-chemical properties including stability, should be known prior to the initiation of any toxicity study.

The physico-chemical properties of the test substance provide important information for the selection of the route of administration, the design of each particular study and the handling and storage of the test substance.

The development of an analytical method for qualitative and quantitative determination of the test substance (including major impurities when possible) in the dosing medium and the biological material should precede the initiation of the study.

All information relating to the identification, the physico-chemical properties, the purity, and behaviour of the test substance should be included in the test report.

B. ANIMAL CARE

Stringent control of environmental conditions and proper animal care techniques are essential in toxicity testing.

(i) Housing conditions

The environmental conditions in the experimental animal rooms or enclosures should be appropriate to the test species. For rats, mice and guinea pigs, suitable conditions are a room temperature of 22 ^oC ± 3 ^oC with a relative humidity of 30 to 70 %; for rabbits the temperature should be 20 ± 3 ^oC with a relative humidity of 30 to 70 %.

Some experimental techniques are particularly sensitive to temperature effects and, in these cases, details of appropriate conditions are included in the description of the test method. In all investigations of toxic effects, the temperature and humidity should be monitored, recorded, and included in the final report of the study.

Lighting should be artificial, the sequence being 12 hours light, 12 hours dark. Details of the lighting pattern should be recorded and included in the final report of the study.

Unless otherwise specified in the method, animals may be housed individually, or be caged in small groups of the same sex; for group caging, no more than five animals should be housed per cage.

In reports of animal experiments, it is important to indicate the type of caging used and the number of animals housed in each cage both during exposure to the chemical and any subsequent observation period.

(ii) Feeding conditions

Diets should meet all the nutritional requirements of the species under test. Where test substances are administered to animals in their diet the nutritional value may be reduced by interaction between the substance and a dietary constituent. The possibility of such a reaction should be considered when interpreting the results of tests. Conventional laboratory diets may be used with an unlimited supply of drinking water. The choice of the diet may be influenced by the need to ensure a suitable admixture of a test substance when administered by this method.

Dietary contaminants which are known to influence the toxicity should not be present in interfering concentrations.

C. ALTERNATIVE TESTING

The European Union is committed to promoting the development and validation of alternative techniques which can provide the same level of information as current animal tests, but which use fewer animals, cause less suffering or avoid the use of animals completely.

Such methods, as they become available, must be considered wherever possible for hazard characterisation and consequent classification and labelling for intrinsic hazards and chemical safety assessment.

D. EVALUATION AND INTERPRETATION

When tests are evaluated and interpreted, limitations in the extent to which the results of animal and in vitro studies can be extrapolated directly to man must be considered and therefore, evidence of adverse effects in humans, where available, may be used for confirmation of testing results.

E. LITERATURE REFERENCES

Most of these methods are developed within the framework of the OECD programme for Testing Guidelines, and should be performed in conformity with the principles of Good Laboratory Practice, in order to ensure as wide as possible ‘mutual acceptance of data’.

Additional information may be found in the references listed in the OECD guidelines and the relevant literature published elsewhere.

B.1 bis.   ACUTE ORAL TOXICITY — FIXED DOSE PROCEDURE

1. METHOD

This test method is equivalent to OECD TG 420 (2001)

1.1.   INTRODUCTION

Traditional methods for assessing acute toxicity use death of animals as an endpoint. In 1984, a new approach to acute toxicity testing was suggested by the British Toxicology Society based on the administration at a series of fixed dose levels (1). The approach avoided using death of animals as an endpoint, and relied instead on the observation of clear signs of toxicity at one of a series of fixed dose levels. Following UK (2) and international (3) in vivo validation studies the procedure was adopted as a testing method in 1992. Subsequently, the statistical properties of the Fixed Dose Procedure have been evaluated using mathematical models in a series of studies (4)(5)(6). Together, the in vivo and modelling studies have demonstrated that the procedure is reproducible, uses fewer animals and causes less suffering than the traditional methods and is able to rank substances in a similar manner to the other acute toxicity testing methods.

Guidance on the selection of the most appropriate test method for a given purpose can be found in the Guidance Document on Acute Oral Toxicity Testing (7). This guidance document also contains additional information on the conduct and interpretation of Testing Method B.1bis.

It is a principle of the method that in the main study only moderately toxic doses are used, and that administration of doses that are expected to be lethal should be avoided. Also, doses that are known to cause marked pain and distress, due to corrosive or severely irritant actions, need not be administered. Moribund animals, or animals obviously in pain or showing signs of severe and enduring distress shall be humanely killed, and are considered in the interpretation of the test results in the same way as animals that died on test. Criteria for making the decision to kill moribund or severely suffering animals, and guidance on the recognition of predictable or impending death, are the subject of a separate Guidance Document (8).

The method provides information on the hazardous properties and allows the substance to be ranked and classified according to the Globally Harmonised System (GHS) for the classification of chemicals which cause acute toxicity (9).

The testing laboratory should consider all available information on the test substance prior to conducting the study. Such information will include the identity and chemical structure of the substance; its physico-chemical properties; the results of any other in vitro or in vivo toxicity tests on the substance; toxicological data on structurally related substances; and the anticipated use(s) of the substance. This information is necessary to satisfy all concerned that the test is relevant for the protection of human health, and will help in the selection of an appropriate starting dose.

1.2.   DEFINITIONS

Acute oral toxicity: refers to those adverse effects occurring following oral administration of a single dose of a substance or multiple doses given within 24 hours.

Delayed death: means that an animal does not die or appear moribund within 48 hours but dies later during the 14-day observation period.

Dose: is the amount of test substance administered. Dose is expressed as weight of test substance per unit weight of test animal (e.g. mg/kg).

Evident toxicity: is a general term describing clear signs of toxicity following the administration of test substance (see (3) for examples) such that at the next highest fixed dose either severe pain and enduring signs of severe distress, moribund status (criteria are presented in the Humane Endpoints Guidance Document (8)), or probable mortality in most animals can be expected.

GHS: Globally Harmonised Classification System for Chemical Substances and Mixtures. A joint activity of OECD (human health and the environment), UN Committee of Experts on Transport of Dangerous Goods (physical-chemical properties) and ILO (hazard communication) and coordinated by the Interorganisation Programme for the Sound Management of Chemicals (IOMC).

Impending death: when moribund state or death is expected prior to the next planned time of observation. Signs indicative of this state in rodents could include convulsions, lateral position, recumbence and tremor. (See the Humane Endpoint Guidance Document (8) for more details).

LD 50 (median lethal dose): is a statistically derived single dose of a substance that can be expected to cause death in 50 % of animals when administered by the oral route. The LD50 value is expressed in terms of weight of test substance per unit weight of test animal (mg/kg).

Limit dose: refers to a dose at an upper limitation on testing (2 000 or 5 000 mg/kg).

Moribund status: being in a state of dying or inability to survive, even if treated. (See the Humane Endpoint Guidance Document (8) for more details).

Predictable death: presence of clinical signs indicative of death at a known time in the future before the planned end of the experiment, for example: inability to reach water or food. (See the Humane Endpoint Guidance Document (8) for more details).

1.3.   PRINCIPLE OF THE TEST METHOD

Groups of animals of a single sex are dosed in a stepwise procedure using the fixed doses of 5, 50, 300 and 2 000 mg/kg (exceptionally an additional fixed dose of 5 000 mg/kg may be considered, see Section 1.6.2). The initial dose level is selected on the basis of a sighting study as the dose expected to produce some signs of toxicity without causing severe toxic effects or mortality. Clinical signs and conditions associated with pain, suffering, and impending death, are described in detail in a separate OECD Guidance Document (8). Further groups of animals may be dosed at higher or lower fixed doses, depending on the presence or absence of signs of toxicity or mortality. This procedure continues until the dose causing evident toxicity or no more than one death is identified, or when no effects are seen at the highest dose or when deaths occur at the lowest dose.

1.4.   DESCRIPTION OF THE TEST METHOD

1.4.1.   Selection of animal species

The preferred rodent species is the rat, although other rodent species may be used. Normally females are used (7). This is because literature surveys of conventional LD50 tests show that usually there is little difference in sensitivity between the sexes, but in those cases where differences are observed, females are generally slightly more sensitive (10). However, if knowledge of the toxicological or toxicokinetic properties of structurally related chemicals indicates that males are likely to be more sensitive then this sex should be used. When the test is conducted in males, adequate justification should be provided.

Healthy young adult animals of commonly used laboratory strains should be employed. Females should be nulliparous and non-pregnant. Each animal, at the commencement of its dosing, should be between eight and 12 weeks old and its weight should fall in an interval within ± 20 % of the mean weight of any previously dosed animals.

1.4.2.   Housing and feeding conditions

The temperature of the experimental animal room should be 22 ^oC (± 3 ^oC). Although the relative humidity should be at least 30 % and preferably not exceed 70 % other than during room cleaning the aim should be 50-60 %. Lighting should be artificial, the sequence being 12 hours light, 12 hours dark. For feeding, conventional laboratory diets may be used with an unlimited supply of drinking water. Animals may be group-caged by dose, but the number of animals per cage must not interfere with clear observations of each animal.

1.4.3.   Preparation of animals

The animals are randomly selected, marked to permit individual identification, and kept in their cages for at least five days prior to the start of dosing to allow for acclimatisation to the laboratory conditions.

1.4.4.   Preparation of doses

In general test substances should be administered in a constant volume over the range of doses to be tested by varying the concentration of the dosing preparation. Where a liquid end product or mixture is to be tested however, the use of the undiluted test substance, i.e. at a constant concentration, may be more relevant to the subsequent risk assessment of that substance, and is a requirement of some regulatory authorities. In either case, the maximum dose volume for administration must not be exceeded. The maximum volume of liquid that can be administered at one time depends on the size of the test animal. In rodents, the volume should not normally exceed 1ml /100 g of body weight: however in the case of aqueous solutions 2 ml/100 g body weight can be considered. With respect to the formulation of the dosing preparation, the use of an aqueous solution/suspension/emulsion is recommended wherever possible, followed in order of preference by a solution/suspension/emulsion in oil (e.g. corn oil) and then possibly solution in other vehicles. For vehicles other than water the toxicological characteristics of the vehicle should be known. Doses must be prepared shortly prior to administration unless the stability of the preparation over the period during which it will be used is known and shown to be acceptable.

1.5.   PROCEDURE

1.5.1.   Administration of doses

The test substance is administered in a single dose by gavage using a stomach tube or a suitable intubation canula. In the unusual circumstance that a single dose is not possible, the dose may be given in smaller fractions over a period not exceeding 24 hours.

Animals should be fasted prior to dosing (e.g. with the rat, food but not water should be withheld over-night; with the mouse, food but not water should be withheld for three to four hours). Following the period of fasting, the animals should be weighed and the test substance administered. After the substance has been administered, food may be withheld for a further three to four hours in rats or one to two hours in mice. Where a dose is administered in fractions over a period of time, it may be necessary to provide the animals with food and water depending on the length of the period.

1.5.2.   Sighting study