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)

Generally at least three treatment groups and a vehicle control group should be used. The vehicle control group should be treated in a manner identical to the treatment group, except that administration of the test substance is omitted.

Sufficient number of hens should be utilised in each group of birds so that at least six birds can be killed for biochemical determinations (three at each of two timepoints) and six birds can survive the 14-day post-treatment observation period for pathology.

1.4.2.3.   Dose levels

Dose levels should be selected taking into account the results from an acute test on delayed neurotoxicity and any other existing toxicity or kinetic data available for the test compound. The highest dose level should be chosen with the aim of inducing toxic effects, preferably delayed neurotoxicity, but not death nor obvious suffering. Thereafter, a descending sequence of dose levels should be selected with a view to demonstrate any dose-related response and no-observed-adverse effects at the lowest dose level.

1.4.2.4.   Limit test

If a test at a dose level of at least 1 000 mg/kg body weight/day, using the procedures described for this study, produces no observable toxic effects and if toxicity would not be expected based upon data from structurally related substances, then a study using a higher dose may not be considered necessary. The limit test applies except when expected human exposure indicates the need for a higher dose level to be used.

1.4.2.5.   Observation period

All the animals should be observed at least daily during the exposure period and 14 days after, unless scheduled necropsy.

1.4.3.   Procedure

Animals are dosed with the test substance on seven days per week for a period of 28 days.

General observations

Observations should start immediately after treatment begins. All hens should be carefully observed at least once daily on each of the 28 days of treatment, and for 14 days after dosing or until scheduled kill. All signs of toxicity should be recorded including their time of onset, type, severity and duration. Observations should include, but not be limited to, behavioural abnormalities. Ataxia should be measured on an ordinal grading scale consisting of at least four levels, and paralysis should be noted. At least twice a week the hens should be taken outside the cages and subjected to a period of forced motor activity, such as ladder climbing, in order to facilitate the observation of minimal toxic effects. Moribund animals in severe distress or pain should be removed when noticed, humanely killed and necropsied.

Body weight

All hens should be weighed just prior to the first administration of the test substance and at least once a week thereafter.

Biochemistry

Six hens randomly selected from each of the treatment and vehicle control groups should be killed within a few days after the last dose, and the brain and lumbar spinal cord prepared and assayed for neuropathy target esterase (NTE) inhibition activity. In addition, it may also be useful to prepare and assay sciatic nerve tissue for neuropathy target esterase (NTE) inhibition activity. Normally, three birds of the control and each treatment group are killed after 24 hours and three at 48 hours after the last dose. If data from the acute study or other studies (e.g. toxicokinetics) indicate that other times of killing after final dosing are preferable then these times should be used and the rationale documented.

Analyses of acetylcholinesterase (AChE) may also be performed on these samples, if deemed appropriate. However, spontaneous reactivation of AChE may occur in vivo, and so lead to underestimation of the potency of the substance as an AChE inhibitor.

Gross necropsy

Gross necropsy of all animals (scheduled killed and killed when moribund) should include observation of the appearance of the brain and spinal cord.

Histopathological examination

Neural tissue from animals surviving the observation period and not used for biochemical studies should be subjected to microscopic examination. Tissues should be fixed in situ, using perfusion techniques. Sections should include cerebellum (mid longitudinal level), medulla oblongata, spinal cord and peripheral nerves. The spinal cord sections should be taken from the upper cervical segment, the mid-thoracic and the lumbo-sacral regions. Sections of the distal region of the tibial nerve and its branches to the gastrocnemial muscle and of the sciatic nerve should be taken. Sections should be stained with appropriate myelin and axon-specific stains. Initially, microscopic examination should be carried out on the preserved tissues of all animals in the control and high dose group. When there is evidence of effects in the high dose group, microscopic examination should also be carried out in hens from the intermediate and low dose groups.

2. DATA

Negative results on the endpoints selected in this method (biochemistry, histopathology and behavioural observation) would not normally require further testing for delayed neurotoxicity. Equivocal or inconclusive results for these endpoints may require further evaluation.

Individual data should be provided. Additionally, all data should be summarised in tabular form, showing for each test group the number of animals at the start of the test, the number of animals showing lesions, behavioural or biochemical effects, the types and severity of these lesions or effects, and the percentage of animals displaying each type and severity of lesion or effect.

The findings of this study should be evaluated in terms of the incidence, severity, and correlation of behavioural, biochemical and histopathological effects and any other observed effects in each of the treated and control groups.

Numerical results should be evaluated by appropriate and generally acceptable statistical methods. The statistical methods should be selected during the design of the study.

3. REPORTING

TEST REPORT

The test report shall, if possible, include the following information:

• 3.1. Test animals:
• — strain used,
• — number and age of animals,
• — source, housing conditions, etc.,
• — individual weights of animals at the start of the test.

• 3.2. Test conditions:
• — details of test substance preparation, stability and homogeneity, where appropriate,
• — justification for choice of vehicle,
• — details of the administration of the test substance,
• — details of food and water quality,
• — rationale for dose selection,
• — specification of doses administered, including details of the vehicle, volume and physical form of the material administered,
• — rationale for choosing other times for biochemical determination, if other than 24 and 48 h.

• 3.3. Results:
• — body weight data,
• — toxic response data by dose level, including mortality,
• — no-observed adverse effect level,
• — nature, severity and duration of clinic observations (whether reversible or not),
• — a detailed description of biochemical methods and findings,
• — necropsy findings,
• — a detailed description of all histopathological findings,
• — statistical treatment of results, where appropriate.

Discussion of results.

Conclusions.

4. REFERENCES

This method is analogous to OECD TG 419.

B.39.   UNSCHEDULED DNA SYNTHESIS (UDS) TEST WITH MAMMALIAN LIVER CELLS IN VIVO

This test method has been deleted, as it is no longer recognised as being appropriate for generating information on the toxicological properties of chemicals for the purpose of Regulation (EC) No 1907/2006. The applicable test methods for the endpoint in question appear in Table 2 of Part 0.

B.40.   IN VITRO SKIN CORROSION: TRANSCUTANEOUS ELECTRICAL RESISTANCE TEST METHOD (TER)

INTRODUCTION

• 1.This test method (TM) is equivalent to OECD test guideline (TG) 430 (2015). Skin corrosion refers to the production of irreversible damage to the skin manifested as visible necrosis through the epidermis and into the dermis, following the application of a test chemical [as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Chemicals (GHS) (1) and the European Union (EU) Regulation 1272/2008 on Classification, Labelling and Packaging of Substances and Mixtures (CLP) ([^E0017])]. This updated test method B.40 provides an in vitro procedure allowing the identification of non-corrosive and corrosive substances and mixtures in accordance with UN GHS (1) and CLP

• 2.The assessment of skin corrosivity has typically involved the use of laboratory animals (TM B.4, equivalent to OECD TG 404 originally adopted in 1981, and revised in 1992, 2002 and 2015) (2). In addition to the present TM B.40, other in vitro test methods for testing of skin corrosion potential of chemicals have been validated and adopted as TM B.40bis (equivalent to OECD TG 431) (3) and TM B.65 (equivalent to OECD TG 435) (4), that are also able to identify sub-categories of corrosive chemicals when required. Several validated in vitro test methods have been adopted as TM B.46 (equivalent to OECD TG 439 (5), to be used for the testing of skin irritation. An OECD guidance document on Integrated Approaches to Testing and Assessment (IATA) for Skin Corrosion and Irritation describes several modules which group various information sources and analysis tools and provides guidance on (i) how to integrate and use existing testing and non-testing data for the assessment of skin irritation and skin corrosion potentials of chemicals and (ii) proposes an approach when further testing is needed (6).

• 3.This test method addresses the human health endpoint skin corrosion. It is based on the rat skin transcutaneous electrical resistance (TER) test method, which utilises skin discs to identify corrosives by their ability to produce a loss of normal stratum corneum integrity and barrier function. The corresponding OECD test guideline was originally adopted in 2004 and updated in 2015 to refer to the IATA guidance document.

• 4.In order to evaluate in vitro skin corrosion testing for regulatory purposes, pre-validation studies (7) followed by a formal validation study of the rat skin TER test method for assessing skin corrosion were conducted (8) (9) (10) (11). The outcome of these studies led to the recommendation that the TER test method (designated the Validated Reference Method – VRM) could be used for regulatory purposes for the assessment of in vivo skin corrosivity (12) (13) (14).

• 5.Before a proposed similar or modified in vitro TER test method for skin corrosion other than the VRM can be used for regulatory purposes, its reliability, relevance (accuracy), and limitations for its proposed use should be determined to ensure its similarity to the VRM, in accordance with the requirements of the Performance Standards (PS) (15). OECD Mutual Acceptance of Data will only be guaranteed after any proposed new or updated test method following the PS have been reviewed and included in the corresponding OECD test guideline.

DEFINITIONS

• 6.Definitions used are provided in the Appendix.

INITIAL CONSIDERATIONS

• 7.A validation study (10) and other published studies (16) (17) have reported that the rat skin TER test method is able to discriminate between known skin corrosives and non-corrosives with an overall sensitivity of 94 % (51/54) and specificity of 71 % (48/68) for a database of 122 substances.

• 8.This test method addresses in vitro skin corrosion. It allows the identification of non-corrosive and corrosive test chemicals in accordance with the UN GHS/CLP. A limitation of this test method, as demonstrated by the validation studies (8) (9) (10) (11), is that it does not allow the sub-categorisation of corrosive substances and mixtures in accordance with the UN GHS/ CLP. The applicable regulatory framework will determine how this test method will be used. While this test method does not provide adequate information on skin irritation, it should be noted that TM B.46 specifically addresses the health effect skin irritation in vitro (5). For a full evaluation of local skin effects after a single dermal exposure, the OECD Guidance Document on IATA should be consulted (6).

• 9.A wide range of chemicals representing mainly substances has been tested in the validation underlying this test method and the empirical database of the validation study amounted to 60 substances covering a wide range of chemical classes (8) (9). On the basis of the overall data available, the test method is applicable to a wide range of chemical classes and physical states including liquids, semi-solids, solids and waxes. However, since for specific physical states test items with suitable reference data are not readily available, it should be noted that a comparably small number of waxes and corrosive solids were assessed during validation. The liquids may be aqueous or non-aqueous; solids may be soluble or insoluble in water. In cases where evidence can be demonstrated on the non-applicability of the test method to a specific category of substances, the test method should not be used for that specific category of substances. In addition, this test method is assumed to be applicable to mixtures as an extension of its applicability to substances. However, due to the fact that mixtures cover a wide spectrum of categories and composition, and that only limited information is currently available on the testing of mixtures, in cases where evidence can be demonstrated on the non-applicability of the test method to a specific category of mixtures (e.g. following a strategy as proposed by Eskes et al., 2012) (18), the test method should not be used for that specific category of mixtures. Before use of the test method on a mixture for generating data for an intended regulatory purpose, it should be considered whether, and if so why, it may provide adequate results for that purpose. Such considerations are not needed, when there is a regulatory requirement for testing of the mixture. Gases and aerosols have not been assessed yet in validation studies (8) (9). While it is conceivable that these can be tested using the TER test method, the current test method does not allow testing of gases and aerosols.

PRINCIPLE OF THE TEST

• 10.The test chemical is applied for up to 24 hours to the epidermal surfaces of skin discs in a two-compartment test system in which the skin discs function as the separation between the compartments. The skin discs are taken from humanely killed rats aged 28-30 days. Corrosive chemicals are identified by their ability to produce a loss of normal stratum corneum integrity and barrier function, which is measured as a reduction in the TER below a threshold level (16) (see paragraph 32). For rat skin TER, a cut-off value of 5k has been selected based on extensive data for a wide range of substances where the vast majority of values were either clearly well above (often > 10 k), or well below (often < 3 k) this value (16). Generally, test chemicals that are non-corrosive in animals but are irritant or non-irritant do not reduce the TER below this cut-off value. Furthermore, use of other skin preparations or other equipment may alter the cut-off value, necessitating further validation.

• 11.A dye-binding step is incorporated into the test procedure for confirmation testing of positive results in the TER including values around 5 k. The dye-binding step determines if the increase in ionic permeability is due to physical destruction of the stratum corneum. The TER method utilising rat skin has shown to be predictive of in vivo corrosivity in the rabbit assessed under TM B.4 (2).

DEMONSTRATION OF PROFICIENCY

• 12.Prior to routine use of the rat skin TER test method that adheres to this test method, laboratories should demonstrate technical proficiency by correctly classifying the twelve Proficiency Substances recommended in Table 1. In situations where a listed substance is unavailable or where justifiable, another substance for which adequate in vivo and in vitro reference data are available may be used (e.g. from the list of reference chemicals (16)) provided that the same selection criteria as described in Table 1 is applied.

Table 1

List of Proficiency Substances [^E0018]

Substance	CASRN	Chemical Class [^E0019]	UN GHS/CLP Cat. Based on In Vivo Results [^E0020]	VRM Cat. Based on In Vitro Results	Physical State	pH [^E0021]
In Vivo Corrosives	In Vivo Corrosives	In Vivo Corrosives	In Vivo Corrosives	In Vivo Corrosives	In Vivo Corrosives	In Vivo Corrosives
N,N’-Dimethyldipropylenetriamine	10563-29-8	organic base	1A	6 × C	L	8,3
1,2-Diaminopropane	78-90-0	organic base	1A	6 × C	L	8,3
Sulfuric acid (10 %)	7664-93-9	inorganic acid	(1A/)1B/1C	5 × C 1 × NC	L	1,2
Potassium hydroxide (10 % aq.)	1310-58-3	inorganic base	(1A/)1B/1C	6 × C	L	13,2
Octanoic (Caprylic) acid	124-07-2	organic acid	1B/1C	4 × C 2 × NC	L	3,6
2-tert-Butylphenol	88-18-6	phenol	1B/1C	4 × C 2 × NC	L	3,9
In Vivo Non-corrosives	In Vivo Non-corrosives	In Vivo Non-corrosives	In Vivo Non-corrosives	In Vivo Non-corrosives	In Vivo Non-corrosives	In Vivo Non-corrosives
Isostearic acid	2724-58-5	organic acid	NC	6 × NC	L	3,6
4-Amino-1,2,4-triazole	584-13-4	organic base	NC	6 × NC	S	5,5
Phenethyl bromide	103-63-9	electrophile	NC	6 × NC	L	3,6
4-(Methylthio)-benzaldehyde	3446-89-7	electrophile	NC	6 × NC	L	6,8
1,9-Decadiene	1647-16-1	neutral organic	NC	6 × NC	L	3,9
Tetrachloroethylene	127-18-4	neutral organic	NC	6 × NC	L	4,5
Abbreviations: aq = aqueous; CASRN = Chemical Abstracts Service Registry Number; VRM = Validated Reference Method; C = corrosive; NC = not corrosive.	Abbreviations: aq = aqueous; CASRN = Chemical Abstracts Service Registry Number; VRM = Validated Reference Method; C = corrosive; NC = not corrosive.	Abbreviations: aq = aqueous; CASRN = Chemical Abstracts Service Registry Number; VRM = Validated Reference Method; C = corrosive; NC = not corrosive.	Abbreviations: aq = aqueous; CASRN = Chemical Abstracts Service Registry Number; VRM = Validated Reference Method; C = corrosive; NC = not corrosive.	Abbreviations: aq = aqueous; CASRN = Chemical Abstracts Service Registry Number; VRM = Validated Reference Method; C = corrosive; NC = not corrosive.	Abbreviations: aq = aqueous; CASRN = Chemical Abstracts Service Registry Number; VRM = Validated Reference Method; C = corrosive; NC = not corrosive.	Abbreviations: aq = aqueous; CASRN = Chemical Abstracts Service Registry Number; VRM = Validated Reference Method; C = corrosive; NC = not corrosive.

The proficiency substances, sorted first by corrosives versus non-corrosives, then by corrosive subcategory and then by chemical class, were selected from the substances used in the ECVAM validation study of the rat skin TER test method (8) (9). Unless otherwise indicated, the substances were tested at the purity level obtained when purchased from a commercial source (8). The selection included, to the extent possible, substances that: (i) are representative of the range of corrosivity responses (e.g. non-corrosives; weak to strong corrosives) that the VRM is capable of measuring or predicting; (ii) are representative of the chemical classes used in the validation study; (iii) reflect the performance characteristics of the VRM; (iv) have chemical structures that are well-defined; (v) induce definitive results in the in vivo reference test method; (vi) are commercially available; and (vii) are not associated with prohibitive disposal costs.

Chemical class assigned by Barratt et al. (8).

The corresponding UN Packing groups are I, II and III, respectively, for the UN GHS/CLP categories 1A, 1B and 1C.

The pH values were obtained from Fentem et al. (9) and Barratt et al. (8).

PROCEDURE

• 13.Standard Operating Procedures (SOP) for the rat skin TER skin corrosion test method are available (19). The rat skin TER test methods covered by this test method should comply with the following conditions:

Animals

• 14.Rats should be used because the sensitivity of their skin to substances in this test method has been previously demonstrated (12) and is the only skin source that has been formally validated (8) (9). The age (when the skin is collected) and strain of the rat is particularly important to ensure that the hair follicles are in the dormant phase before adult hair growth begins.

• 15.The dorsal and flank hair from young, approximately 22 day-old, male or female rats (Wistar-derived or a comparable strain), is carefully removed with small clippers. Then, the animals are washed by careful wiping, whilst submerging the clipped area in antibiotic solution (containing, for example, streptomycin, penicillin, chloramphenicol, and amphotericin, at concentrations effective in inhibiting bacterial growth). Animals are washed with antibiotics again on the third or fourth day after the first wash and are used within 3 days of the second wash, when the stratum corneum has recovered from the hair removal.

Preparation of the skin discs

• 16.Animals are humanely killed when 28-30 days old; this age is critical. The dorso-lateral skin of each animal is then removed and stripped of excess subcutaneous fat by carefully peeling it away from the skin. Skin discs, with a diameter of approximately 20-mm each, are removed. The skin may be stored before discs are used where it is shown that positive and negative control data are equivalent to that obtained with fresh skin.

• 17.Each skin disc is placed over one of the ends of a PTFE (polytetrafluoroethylene) tube, ensuring that the epidermal surface is in contact with the tube. A rubber ‘O’ ring is press-fitted over the end of the tube to hold the skin in place and excess tissue is trimmed away. The rubber ‘O’ ring is then carefully sealed to the end of the PTFE tube with petroleum jelly. The tube is supported by a spring clip inside a receptor chamber containing MgSO4 solution (154 mM) (Figure 1). The skin disc should be fully submerged in the MgSO4 solution. As many as 10-15 skin discs can be obtained from a single rat skin. Tube and ‘O’ ring dimensions are shown in Figure 2.

• 18.Before testing begins, the TER of two skin discs are measured as a quality control procedure for each animal skin. Both discs should give electrical resistance values greater than 10 k for the remainder of the discs to be used for the test method. If the resistance value is less than 10 k, the remaining discs from that skin should be discarded.

Application of the test chemical and control substances

• 19.Concurrent positive and negative controls should be used for each run (experiment) to ensure adequate performance of the experimental model. Skin discs from a single animal should be used in each run (experiment). The suggested positive and negative control test chemicals are 10 M hydrochloric acid and distilled water, respectively.

• 20.Liquid test chemicals (150 μl) are applied uniformly to the epidermal surface inside the tube. When testing solid materials, a sufficient amount of the solid is applied evenly to the disc to ensure that the whole surface of the epidermis is covered. Deionised water (150 μl) is added on top of the solid and the tube is gently agitated. In order to achieve maximum contact with the skin, solids may need to be warmed to 30° C to melt or soften the test chemical, or ground to produce a granular material or powder.

• 21.Three skin discs are used for each test and control chemical in each testing run (experiment). Test chemicals are applied for 24 hours at 20-23° C. The test chemical is removed by washing with a jet of tap water at up to room temperature until no further material can be removed.

TER measurements

• 22.The skin impedance is measured as TER by using a low-voltage, alternating current Wheatstone bridge (18). General specifications of the bridge are 1-3 Volt operating voltage, a sinus or rectangular shaped alternating current of 50 - 1 000 Hz, and a measuring range of at least 0,1-30 k. The databridge used in the validation study measured inductance, capacitance and resistance up to values of 2 000 H, 2 000  F, and 2 M, respectively at frequencies of 100Hz or 1kHz, using series or parallel values. For the purposes of the TER corrosivity assay measurements are recorded in resistance, at a frequency of 100 Hz and using series values. Prior to measuring the electrical resistance, the surface tension of the skin is reduced by adding a sufficient volume of 70 % ethanol to cover the epidermis. After a few seconds, the ethanol is removed from the tube and the tissue is then hydrated by the addition of 3 ml MgSO4 solution (154mM). The databridge electrodes are placed on either side of the skin disc to measure the resistance in kΩ/skin disc (Figure 1). Electrode dimensions and the length of the electrode exposed below the crocodile clips are shown in Figure 2. The clip attached to the inner electrode is rested on the top of the PTFE tube during resistance measurement to ensure that a consistent length of electrode is submerged in the MgSO4 solution. The outer electrode is positioned inside the receptor chamber so that it rests on the bottom of the chamber. The distance between the spring clip and the bottom of the PTFE tube is maintained as a constant (Figure 2), because this distance affects the resistance value obtained. Consequently, the distance between the inner electrode and the skin disc should be constant and minimal (1-2 mm).

• 23.If the measured resistance value is greater than 20 k, this may be due to the remains of the test chemical coating the epidermal surface of the skin disc. Further removal of this coating can be attempted, for example, by sealing the PTFE tube with a gloved thumb and shaking it for approximately 10 seconds; the MgSO4 solution is discarded and the resistance measurement is repeated with fresh MgSO4.

• 24.The properties and dimensions of the test apparatus and the experimental procedure used may influence the TER values obtained. The 5 k corrosive threshold was developed from data obtained with the specific apparatus and procedure described in this test method. Different threshold and control values may apply if the test conditions are altered or a different apparatus is used. Therefore, it is necessary to calibrate the methodology and resistance threshold values by testing a series of Proficiency Substances chosen from the substances used in the validation study (8) (9), or from similar chemical classes to the substances being investigated. A set of suitable Proficiency Substances is identified in Table 1.

Dye Binding Methods

• 25.Exposure of certain non-corrosive materials can result in a reduction of resistance below the cut-off of 5 kΩ allowing the passage of ions through the stratum corneum, thereby reducing the electrical resistance (9). For example, neutral organics and substances that have surface-active properties (including detergents, emulsifiers and other surfactants) can remove skin lipids making the barrier more permeable to ions. Thus, if TER values produced by such chemicals are less than or around 5 kΩ in the absence of visually perceptible damage of the skin discs, an assessment of dye penetration should be carried out on the control and treated tissues to determine if the TER values obtained were the result of increased skin permeability, or skin corrosion (7) (9). In case of the latter where the stratum corneum is disrupted, the dye sulforhodamine B, when applied to the skin surface rapidly penetrates and stains the underlying tissue. This particular dye is stable to a wide range of substances and is not affected by the extraction procedure described below.

Sulforhodamine B dye application and removal

• 26.Following TER assessment, the magnesium sulphate is discarded from the tube and the skin is carefully examined for obvious damage. If there is no obvious major damage (e.g. perforation), 150 l of a 10 % (w/v) dilution in distilled water of the dye sulforhodamine B (Acid Red 52; C.I. 45100; CAS number 3520-42-1), is applied to the epidermal surface of each skin disc for 2 hours. These skin discs are then washed with tap water at up to room temperature for approximately 10 seconds to remove any excess/unbound dye. Each skin disc is carefully removed from the PTFE tube and placed in a vial (e.g. a 20-ml glass scintillation vial) containing deionised water (8 ml). The vials are agitated gently for 5 minutes to remove any additional unbounddye. This rinsing procedure is then repeated, after which the skin discs are removed and placed into vials containing 5ml of 30 % (w/v) sodium dodecyl sulphate (SDS) in distilled water and are incubated overnight at 60° C.

• 27.After incubation, each skin disc is removed and discarded and the remaining solution is centrifuged for 8 minutes at 21° C (relative centrifugal force ~175 × g). A 1ml sample of the supernatant is diluted 1 in 5 (v/v) [i.e. 1ml + 4ml] with 30 % (w/v) SDS in distilled water. The optical density (OD) of the solution is measured at 565 nm.

Calculation of dye content

• 28.The sulforhodamine B dye content per disc is calculated from the OD values (9) (sulforhodamine B dye molar extinction coefficient at 565nm = 8,7 × l0⁴; molecular weight = 580). The dye content is determined for each skin disc by the use of an appropriate calibration curve and mean dye content is then calculated for the replicates.

Acceptability Criteria

• 29.The mean TER results are accepted if the concurrent positive and negative control values fall within the acceptable ranges for the method in the testing laboratory. The acceptable resistance ranges for the methodology and apparatus described above are given in the following table:

Control	Substance	Resistance range (k)
Positive	10 M Hydrochloric acid	0,5 – 1,0
Negative	Distilled water	10 – 25

• 30.The mean dye binding results are accepted on condition that concurrent control values fall within the acceptable ranges for the method. Suggested acceptable dye content ranges for the control substances for the methodology and apparatus described above are given in the following table:

Control	Substance	Dye content range (g/disc)
Positive	10 M Hydrochloric acid	40 – 100
Negative	Distilled water	15 – 35

Interpretation of results

• 31.The cut-off TER value distinguishing corrosive from non-corrosive test chemicals was established during test method optimisation, tested during a pre-validation phase, and confirmed in a formal validation study.

• 32.The prediction model for rat skin TER skin corrosion test method (9) (19), associated with the UN GHS/CLP classification system, is given below:

• The test chemical is considered to be non-corrosive to skin:

• i) if the mean TER value obtained for the test chemical is greater than (>) 5 kΩ, or

• ii) the mean TER value obtained for the test chemical is less than or equal to (≤) 5 kΩ, and
• — the skin discs show no obvious damage(e.g. perforation), and
• — the mean disc dye content is less than (<) the mean disc dye content of the 10 M HCl positive control obtained concurrently (see paragraph 30 for positive control values).

• The test chemical is considered to be corrosive to skin:

• i) if the mean TER value obtained for the test chemical is less than or equal to (≤) 5 kΩ and the skin discs are obviously damaged(e.g. perforated), or

• ii) the mean TER value obtained for the test chemical is less than or equal to (≤) 5 kΩ, and
• — the skin discs show no obvious damage(e.g. perforation), but
• — the mean disc dye content is greater than or equal to (≥) the mean disc dye content of the 10 M HCl positive control obtained concurrently (see paragraph 30 for positive control values).

• 33.A testing run (experiment) composed of at least three replicate skin discs should be sufficient for a test chemical when the classification is unequivocal. However, in cases of borderline results, such as non-concordant replicate measurements and/or mean TER equal to 5 ± 0.5 kΩ, a second independent testing run (experiment) should be considered, as well as a third one in case of discordant results between the first two testing runs (experiments).

DATA AND REPORTING

Data

• 34.Resistance values (kΩ) and dye content values (μg/disc), where appropriate, for the test chemical, as well as for positive and negative controls should be reported in tabular form, including data for each individual replicate disc in each testing run (experiment) and mean values ± SD. All repeat experiments should be reported. Observed damage in the skin discs should be reported for each test chemical.

Test report

• 35.The test report should include the following information:

Test Chemical and Control Substances:

• — Mono-constituent substance: chemical identification, such as IUPAC or CAS name, CAS number, SMILES or InChI code, structural formula, purity, chemical identity of impurities as appropriate and practically feasible, etc;

• — Multi-constituent substance, UVCB and mixture: characterised as far as possible by chemical identity (see above), quantitative occurrence and relevant physico-chemical properties of the constituents;

• — Physical appearance, water solubility, and additional relevant physico-chemical properties;

• — Source, lot number if available;

• — Treatment of the test chemical/control substance prior to testing, if applicable (e.g. warming, grinding);

• — Stability of the test chemical, limit date for use, or date for re-analysis if known;

• — Storage conditions.

Test Animals:

• — Strain and sex used;

• — Age of the animals when used as donor animals;

• — Source, housing condition, diet, etc.;

• — Details of the skin preparation.

Test Conditions:

• — Calibration curves for test apparatus;

• — Calibration curves for dye binding test performance, band pass used for measuring OD values, and OD linearity range of measuring device (e.g. spectrophotometer), if appropriate;

• — Details of the test procedure used for TER measurements;

• — Details of the test procedure used for the dye binding assessment, if appropriate;

• — Test doses used, duration of exposure period(s) and temperature(s) of exposure;

• — Details on washing procedure used after the exposure period;

• — Number of replicate skin discs used per test chemical and controls (positive and negative control);

• — Description of any modification of the test procedure;

• — Reference to historical data of the model. This should include, but is not limited to:

ii) Acceptability of the positive and negative control dye content values (in μg/disc) with reference to positive and negative control dye content ranges iii) Acceptability of the test results with reference to historical variability between skin disc replicates

• — Description of decision criteria/prediction model applied.

Results:

• — Tabulation of data from the TER and dye binding assays (if appropriate) for individual test chemicals and controls, for each testing run (experiment) and each skin disc replicate (individual animals and individual skin samples), means, SDs and CVs;

• — Description of any effects observed;

• — The derived classification with reference to the prediction model/decision criteria used.

Discussion of the results

Conclusions

LITERATURE

• (1) United Nations (UN) (2013). Globally Harmonized System of Classification and Labelling of Chemicals (GHS), Second Revised Edition, UN New York and Geneva, 2013. Available at: [http://www.unece.org/trans/danger/publi/ghs/ghs_rev05/05files_e.html].

• (2) Chapter B.4 of this Annex, Acute Dermal Irritation, Corrosion.

• (3) Chapter B.40bis of this Annex, In Vitro Skin Model.

• (4) Chapter B.65 of this Annex, In Vitro Membrane Barrier Test Method.

• (5) Chapter B.46 of this Annex, In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method.

• (6) OECD (2014). Guidance document on Integrated Approaches to Testing and Assessment for Skin Irritation/Corrosion. Environment, Health and Safety Publications, Series on Testing and Assessment, (No 203), Organisation for Economic Cooperation and Development, Paris.

• (7) Botham P.A., Chamberlain M., Barratt M.D., Curren R.D., Esdaile D.J., Gardner J.R., Gordon V.C., Hildebrand B., Lewis R.W., Liebsch M., Logemann P., Osborne R., Ponec M., Regnier J.F., Steiling W., Walker A.P., and Balls M. (1995). A Prevalidation Study on In Vitro Skin Corrosivity Testing. The Report and Recommendations of ECVAM Workshop 6.ATLA 23, 219-255.

• (8) Barratt M.D., Brantom P.G., Fentem J.H., Gerner I., Walker A.P., and Worth A.P. (1998). The ECVAM International Validation Study on In Vitro Tests for Skin Corrosivity. 1. Selection and Distribution of the Test Chemicals. Toxic.In Vitro 12, 471-482.

• (9) Fentem J.H., Archer G.E.B., Balls M., Botham P.A., Curren R.D., Earl L.K., Esdaile D.J., Holzhütter H.-G., and Liebsch M. (1998). The ECVAM International Validation Study on In Vitro Tests For Skin Corrosivity. 2. Results and Evaluation by the Management Team. Toxic.In Vitro12, 483- 524.

• (10) Balls M., Blaauboer B.J., Fentem J.H., Bruner L., Combes R.D., Ekwall B., Fielder R.J., Guillouzo A., Lewis R.W., Lovell D.P., Reinhardt C.A., Repetto G., Sladowski D., Spielmann H., and Zucco F. (1995). Practical Aspects of the Validation of Toxicity Test Procedures. The Report and Recommendations of ECVAM Workshops.ATLA23, 129-147.

• (11) ICCVAM (Interagency Coordinating Committee on the Validation of Alternative Methods). (1997). Validation and Regulatory Acceptance of Toxicological Test Methods. NIH Publication No 97-3981. National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA.

• (12) EC-ECVAM (1998). Statement on the Scientific Validity of the Rat Skin Transcutaneos Electrical Resistance (TER) Test (an In Vitro Test for Skin Corrosivity), Issued by the ECVAM Scientific Advisory Committee (ESAC10), 3 April 1998.

• (13) ECVAM (1998). ECVAM News & Views. ATLA 26, 275-280.

• (14) ICCVAM (Interagency Coordinating Committee on the Validation of Alternative Methods) (2002). ICCVAM Evaluation of EpiDerm^TM (EPI-200), EPISKIN^TM (SM), and the Rat Skin Transcutaneous Electrical Resistance (TER) Assay: In Vitro Test Methods for Assessing Dermal Corrosivity Potential of Chemicals. NIH Publication No 02-4502. National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA.

• (15) OECD (2015). Performance Standards for the Assessment of Proposed Similar or Modified In Vitro Transcutaneous Electrical Resistance (TER) Test Method for Skin Corrosion in Relation to TG 430. Environmental Health and Safety Publications, Series on Testing and Assessment No 218. Organisation for Economic Cooperation and Development, Paris.

• (16) Oliver G.J.A., Pemberton M.A., and Rhodes C. (1986). An In Vitro Skin Corrosivity Test -Modifications and Validation. Fd. Chem. Toxicol.24, 507-512.

• (17) Botham P.A., Hall T.J., Dennett R., McCall J.C., Basketter D.A., Whittle E., Cheeseman M., Esdaile D.J., and Gardner J. (1992). The Skin Corrosivity Test In Vitro: Results of an Interlaboratory Trial. Toxicol. In Vitro 6,191-194.

• (18) Eskes C., Detappe V., Koëter H., Kreysa J., Liebsch M., Zuang V., Amcoff P., Barroso J., Cotovio J., Guest R., Hermann M., Hoffmann S., Masson P., Alépée N., Arce L.A., Brüschweiler B., Catone T., Cihak R., Clouzeau J., D'Abrosca F., Delveaux C., Derouette J.P., Engelking O., Facchini D., Fröhlicher M., Hofmann M., Hopf N., Molinari J., Oberli A., Ott M., Peter R., Sá-Rocha V.M., Schenk D., Tomicic C., Vanparys P., Verdon B., Wallenhorst T., Winkler G.C. and Depallens O. (2012). Regulatory Assessment of In Vitro Skin Corrosion and Irritation Data Within the European Framework: Workshop Recommendations. Regul.Toxicol.Pharmacol. 62, 393-403.

• (19) TER SOP (December 2008). INVITTOX Protocol (No 115) Rat Skin Transcutaneous Electrical Resistance (TER) Test.

• (20) OECD (2005). Guidance Document on the Validation and International Acceptance of New or Updated Test Methods for Hazard Assessment. Environment, Health and Safety Publications, Series on Testing and Assessment (No 34), Organisation for Economic Cooperation and Devlopment, Paris.

Figure 1

Apparatus for the Rat Skin Ter Assay

Image omitted; see official source

Figure 2

Dimensions Of The Polytetrafluoroethylene (Ptfe) And Receptor Tubes And Electrodes Used

Image omitted; see official source

Critical factors of the apparatus shown above:

• — The inner diameter of the PTFE tube,

• — The length of the electrodes relative to the PTFE tube and receptor tube, such that the skin disc should not be touched by the electrodes and that a standard length of electrode is in contact with the MgSO4 solution,

• — The amount of MgSO4 solution in the receptor tube should give a depth of liquid, relative to the level in the PTFE tube, as shown in Figure 1,

• — The skin disc should be fixed well enough to the PTFE tube, such that the electrical resistance is a true measure of the skin properties.

Appendix

DEFINITIONS

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 (20).

C : Corrosive.

Chemical : A substance or a mixture.

Concordance : A measure of test method performance for test methods that give a categorical result, and is one aspect of relevance. The term is sometimes used interchangeably with accuracy, and is defined as the proportion of all chemicals tested that are correctly classified as positive or negative. Concordance is highly dependent on the prevalence of positives in the types of test chemical being examined (20).

GHS (Globally Harmonized System of Classification and Labelling of Chemicals (UN)) : A system proposing the classification of chemicals (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).

IATA : Integrated Approach on Testing and Assessment.

Mixture : A mixture or solution composed of two or more substances.

Mono-constituent substance : A substance, defined by its quantitative composition, in which one main constituent is present to at least 80 % (w/w).

Multi-constituent substance : A substance, defined by its quantitative composition, in which more than one main constituent is present in a concentration ≥ 10 % (w/w) and < 80 % (w/w). A multi-constituent substance is the result of a manufacturing process. The difference between mixture and multi-constituent substance is that a mixture is obtained by blending of two or more substances without chemical reaction. A multi-constituent substance is the result of a chemical reaction.

NC : Non corrosive.

OD : Optical Density.

PC : Positive Control, a replicate containing all components of a test system and treated with a substance known to induce a positive response. To ensure that variability in the positive control response across time can be assessed, the magnitude of the positive response should not be excessive.

Performance standards (PS) : Standards, based on a validated test 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 Chemicals selected from among the chemicals used to demonstrate the acceptable performance of the validated test method; and (iii) the similar levels of reliability and accuracy, based on what was obtained for the validated test method, that the proposed test method should demonstrate when evaluated using the minimum list of Reference Chemicals.

Relevance : Description of relationship of the test method to the effect of interest and whether it is meaningful and useful for a particular purpose. It is the extent to which the test method correctly measures or predicts the biological effect of interest. Relevance incorporates consideration of the accuracy (concordance) of a test method (20).

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 (20).

Sensitivity : The proportion of all positive/active chemicals that are correctly classified by the test method. 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 (20).

Skin corrosion in vivo : The production of irreversible damage of the skin; namely, visible necrosis through the epidermis and into the dermis, following the application of a test chemical for up to four hours. Corrosive reactions are typified by ulcers, bleeding, bloody scabs, and, by the end of observation at 14 days, by discoloration due to blanching of the skin, complete areas of alopecia, and scars. Histopathology should be considered to evaluate questionable lesions.

Specificity : The proportion of all negative/inactive chemicals that are correctly classified by the test method. 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 (20).

Substance : A chemical element and its compounds in the natural state or obtained by any production process, including any additive necessary to preserve its stability and any impurities deriving from the process used, but excluding any solvent which may be separated without affecting the stability of the substance or changing it composition.

(Testing) run : A single test chemical concurrently tested in a minimum of three replicate skin discs.

Test chemical : Any substance or mixture tested using this test method.

Transcutaneous Electrical Resistance (TER) : is a measure of the electrical impedance of the skin, as a resistance value in kilo Ohms. A simple and robust method of assessing barrier function by recording the passage of ions through the skin using a Wheatstone bridge apparatus.

UVCB : Substances of unknown or variable composition, complex reaction products or biological materials.

B.40bis.   IN VITRO SKIN CORROSION: RECONSTRUCTED HUMAN EPIDERMIS (RhE) TEST METHOD

The full description of this test method has been deleted. The equivalent international test method appears in Table 2 of Part 0.

B.41.   IN VITRO 3T3 NRU PHOTOTOXICITY TEST

The full description of this test method has been deleted. The equivalent international test method appears in Table 2 of Part 0.

B.42.   SKIN SENSITISATION: LOCAL LYMPH NODE ASSAY

INTRODUCTION

• 1.OECD Guidelines for the Testing of Chemicals and EU Test Methods based on them are periodically reviewed in light of scientific progress, changing regulatory needs, and animal welfare considerations. The original Test Method (TM) for the determination of skin sensitisation in the mouse, the Local Lymph Node Assay (LLNA; OECD Test Guideline 429; Chapter B.42 of this Annex) was adopted previously (1). The details of the validation of the LLNA and a review of the associated work have been published (2) (3) (4) (5) (6) (7) (8) (9) (10) (11). The updated LLNA is based on the evaluation of experience and scientific data (12). This is the second TM to be designed for assessing skin sensitisation potential of chemicals (substances and mixtures) in animals. The other TM (i.e. OECD Test Guideline 406; Chapter B.6 of this Annex) utilises guinea pig tests, notably the guinea pig maximisation test and the Buehler test (13). The LLNA provides advantages over B.6 and OECD Test Guideline 406 (13) with regard to animal welfare. This updated LLNA TM includes a set of Performance Standards (PS) (Appendix 1) that can be used to evaluate the validation status of new and/or modified test methods that are functionally and mechanistically similar to the LLNA, in accordance with the principles of OECD Guidance Document No 34 (14).

• 2.The LLNA studies the induction phase of skin sensitisation and provides quantitative data suitable for dose-response assessment. It should be noted that the mild/moderate sensitisers which are recommended as suitable positive control chemicals (PC) for guinea pig test methods (i.e. B.6; OECD Test Guideline 406) (13) are also appropriate for use with the LLNA (6) (8) (15). A reduced LLNA (rLLNA) approach, which could use up to 40 % fewer animals is also described as an option in this TM (16) (17) (18). The rLLNA may be used when there is a regulatory need to confirm a negative prediction of skin sensitising potential, provided there is adherence to all other LLNA protocol specifications, as described in this TM. Prediction of a negative outcome should be made based on all available information as described in paragraph 4. Before applying the rLLNA approach, clear justifications and scientific rationale for its use should be provided. If, against expectations, a positive or equivocal result is obtained in the rLLNA, additional testing may be needed in order to interpret or clarify the finding. The rLLNA should not be used for the hazard identification of skin sensitising test substances when dose-response information is needed such as sub-categorisation for Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures and UN Globally Harmonised System of Classification and Labelling of Chemicals.

DEFINITIONS

• 3.Definitions used are provided in Appendix 2.

INITIAL CONSIDERATIONS AND LIMITATIONS

• 4.The LLNA provides an alternative method for identifying potential skin sensitising chemicals. This does not necessarily imply that in all instances the LLNA should be used in place of guinea pig tests (i.e. B.6; OECD Test Guideline 406) (13), but rather that the assay is of equal merit and may be employed as an alternative in which positive and negative results generally no longer require further confirmation. 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 test substance; its physicochemical properties; the results of any other in vitro or in vivo toxicity tests on the test substance; and toxicological data on structurally related chemicals. This information should be considered in order to determine whether the LLNA is appropriate for the substance (given the incompatibility of limited types of chemicals with the LLNA — see paragraph 5) and to aid in dose selection.

• 5.The LLNA is an in vivo method and, as a consequence, will not eliminate the use of animals in the assessment of allergic contact sensitising activity. It has, however, the potential to reduce the number of animals required for this purpose. Moreover, the LLNA offers a substantial refinement (less pain and distress) of the way in which animals are used for allergic contact sensitisation testing. The LLNA is based upon consideration of immunological events stimulated by chemicals during the induction phase of sensitisation. Unlike guinea pig tests (i.e. B.6; OECD Test Guideline 406) (13) the LLNA does not require that challenge-induced dermal hypersensitivity reactions be elicited. Furthermore, the LLNA does not require the use of an adjuvant, as is the case for the guinea pig maximisation test (13). Thus, the LLNA reduces animal pain and distress. Despite the advantages of the LLNA over B.6 and OECD Test Guideline 406, it should be recognised that there are certain limitations that may necessitate the use of B.6 or OECD Test Guideline 406 (13) (e.g. false negative findings in the LLNA with certain metals, false positive findings with certain skin irritants (such as some surfactant type chemicals) (19) (20), or solubility of the test substance). In addition, chemical classes or substances containing functional groups shown to act as potential confounders (21) may necessitate the use of guinea pig tests (i.e. B.6; OECD Test Guideline 406) (13). Further, based on the limited validation database, which consisted primarily of pesticide formulations, the LLNA is more likely than the guinea pig test to yield a positive result for these types of test substances (22). However, when testing formulations, one could consider including similar substances with known results as benchmark substances to demonstrate that the LLNA is functioning properly (see paragraph 16). Other than such identified limitations, the LLNA should be applicable for testing any substances unless there are properties associated with these substances that may interfere with the accuracy of the LLNA.

PRINCIPLE OF THE TEST

• 6.The basic principle underlying the LLNA is that sensitisers induce proliferation of lymphocytes in the lymph nodes draining the site of test substance application. This proliferation is proportional to the dose and to the potency of the applied allergen and provides a simple means of obtaining a quantitative measurement of sensitisation. Proliferation is measured by comparing the mean proliferation in each test group to the mean proliferation in the vehicle treated control (VC) group. The ratio of the mean proliferation in each treated group to that in the concurrent VC group, termed the Stimulation Index (SI), is determined, and should be ≥ 3 before classification of the test substance as a potential skin sensitiser is warranted. The procedures described here are based on the use of in vivo radioactive labelling to measure an increased number of proliferating cells in the draining auricular lymph nodes. However, other endpoints for assessment of the number of proliferating cells may be employed provided the PS requirements are fully met (Appendix 1).

DESCRIPTION OF THE ASSAY

Selection of animal species

• 7.The mouse is the species of choice for this test. Young adult female mice of CBA/Ca or CBA/J strain, which are nulliparous and non-pregnant, are used. At the start of the study, animals should be between 8-12 weeks old, and the weight variation of the animals should be minimal and not exceed 20 % of the mean weight. Alternatively, other strains and males may be used when sufficient data are generated to demonstrate that significant strain and/or gender-specific differences in the LLNA response do not exist.

Housing and feeding conditions

• 8.Mice should be group-housed (23), unless adequate scientific rationale for housing mice individually is provided. The temperature of the experimental animal room should be 22 ± 3 °C. 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.

Preparation of animals

• 9.The animals are randomly selected, marked to permit individual identification (but not by any form of ear marking), and kept in their cages for at least five days prior to the start of dosing to allow for acclimatisation to the laboratory conditions. Prior to the start of treatment all animals are examined to ensure that they have no observable skin lesions.

Preparation of dosing solutions

• 10.Solid chemicals should be dissolved or suspended in solvents/vehicles and diluted, if appropriate, prior to application to an ear of the mice. Liquid chemicals may be applied neat or diluted prior to dosing. Insoluble chemicals, such as those generally seen in medical devices, should be subjected to an exaggerated extraction in an appropriate solvent to reveal all extractable constituents for testing prior to application to an ear of the mice. Test substances should be prepared daily unless stability data demonstrate the acceptability of storage.

Reliability check

• 11.Positive control chemicals (PC) are used to demonstrate appropriate performance of the assay by responding with adequate and reproducible sensitivity as a sensitising test substance for which the magnitude of the response is well characterised. Inclusion of a concurrent PC is recommended because it demonstrates competency of the laboratory to successfully conduct each assay and allows for an assessment of intra- and inter-laboratory reproducibility and comparability. A PC for each study is also required by some regulatory authorities and therefore users are encouraged to consult the relevant authorities prior to conducting the LLNA. Accordingly, the routine use of a concurrent PC is encouraged to avoid the need for additional animal testing to meet such requirements that might arise from the use of a periodic PC (see paragraph 12). The PC should produce a positive LLNA response at an exposure level expected to give an increase in the SI > 3 over the negative control (NC) group. The PC dose should be chosen such that it does not cause excessive skin irritation or systemic toxicity and the induction is reproducible but not excessive (i.e. a SI > 20 would be excessive). Preferred PC are 25 % hexyl cinnamic aldehyde (Chemical Abstracts Service (CAS) No 101-86-0) in acetone: olive oil (4:1, v/v) and 5 % mercaptobenzothiazole (CAS No 149-30-4) in N,N-dimethylformamide (see Appendix 1, Table 1). There may be circumstances in which, given adequate justification, other PC, meeting the above criteria, may be used.

• 12.While inclusion of a concurrent PC group is recommended, there may be situations in which periodic testing (i.e. at intervals ≤ 6 months) of the PC may be adequate for laboratories that conduct the LLNA regularly (i.e. conduct the LLNA at a frequency of no less than once per month) and have an established historical PC database that demonstrates the laboratory’s ability to obtain reproducible and accurate results with PCs. Adequate proficiency with the LLNA can be successfully demonstrated by generating consistent positive results with the PC in at least 10 independent tests conducted within a reasonable period of time (i.e. less than one year).

• 13.A concurrent PC group should always be included when there is a procedural change to the LLNA (e.g. change in trained personnel, change in test method materials and/or reagents, change in test method equipment, change in source of test animals), and such changes should be documented in laboratory reports. Consideration should be given to the impact of these changes on the adequacy of the previously established historical database in determining the necessity for establishing a new historical database to document consistency in the PC results.

• 14.Investigators should be aware that the decision to conduct a PC study on a periodic basis instead of concurrently has ramifications on the adequacy and acceptability of negative study results generated without a concurrent PC during the interval between each periodic PC study. For example, if a false negative result is obtained in the periodic PC study, negative test substance results obtained in the interval between the last acceptable periodic PC study and the unacceptable periodic PC study may be questioned. Implications of these outcomes should be carefully considered when determining whether to include concurrent PCs or to only conduct periodic PCs. Consideration should also be given to using fewer animals in the concurrent PC group when this is scientifically justified and if the laboratory demonstrates, based on laboratory-specific historical data, that fewer mice can be used (12).

• 15.Although the PC should be tested in the vehicle that is known to elicit a consistent response (e.g. acetone: olive oil; 4:1, v/v), there may be certain regulatory situations in which testing in a non-standard vehicle (clinically/chemically relevant formulation) will also be necessary (24). If the concurrent PC is tested in a different vehicle than the test substance, then a separate VC for the concurrent PC should be included.

• 16.In instances where test substances of a specific chemical class or range of responses are being evaluated, benchmark substances may also be useful to demonstrate that the test method is functioning properly for detecting the skin sensitisation potential of these types of test substances. Appropriate benchmark substances should have the following properties:

• — structural and functional similarity to the class of the test substance being tested;

• — known physical/chemical characteristics;

• — supporting data from the LLNA;

• — supporting data from other animal models and/or from humans.

TEST PROCEDURE

Number of animals and dose levels

• 17.A minimum of four animals is used per dose group, with a minimum of three concentrations of the test substance, plus a concurrent NC group treated only with the vehicle for the test substance, and a PC (concurrent or recent, based on laboratory policy in considering paragraphs 11-15). Testing multiple doses of the PC should be considered, especially when testing the PC on an intermittent basis. Except for absence of treatment with the test substance, animals in the control groups should be handled and treated in a manner identical to that of animals in the treatment groups.

• 18.Dose and vehicle selection should be based on the recommendations given in references (3) and (5). Consecutive doses are normally selected from an appropriate concentration series such as 100 %, 50 %, 25 %, 10 %, 5 %, 2,5 %, 1 %, 0,5 %, etc. Adequate scientific rationale should accompany the selection of the concentration series used. All existing toxicological information (e.g. acute toxicity and dermal irritation) and structural and physicochemical information on the test substance of interest (and/or structurally related substances) should be considered where available, in selecting the three consecutive concentrations so that the highest concentration maximises exposure while avoiding systemic toxicity and/or excessive local skin irritation (3) (25). In the absence of such information, an initial pre-screen test may be necessary (see paragraphs 21-24).

• 19.The vehicle should not interfere with or bias the test result and should be selected on the basis of maximising the solubility in order to obtain the highest concentration achievable while producing a solution/suspension suitable for application of the test substance. Recommended vehicles are acetone: olive oil (4:1, v/v), N,N-dimethylformamide, methyl ethyl ketone, propylene glycol, and dimethyl sulphoxide (19) but others may be used if sufficient scientific rationale is provided. In certain situations it may be necessary to use a clinically relevant solvent or the commercial formulation in which the test substance is marketed as an additional control. Particular care should be taken to ensure that hydrophilic test substances are incorporated into a vehicle system, which wets the skin and does not immediately run off, by incorporation of appropriate solubilisers (e.g. 1 % Pluronic® L92). Thus, wholly aqueous vehicles are to be avoided.

• 20.The processing of lymph nodes from individual mice allows for the assessment of inter-animal variability and a statistical comparison of the difference between test substance and VC group measurements (see paragraph 35). In addition, evaluating the possibility of reducing the number of mice in the PC group is feasible when individual animal data are collected (12). Further, some regulatory authorities require the collection of individual animal data. Nonetheless, pooled animal data may be considered acceptable by some regulatory authorities and in such situations, users may have the option of collecting either individual or pooled animal data.

Pre-screen test

• 21.In the absence of information to determine the highest dose to be tested (see paragraph 18), a pre-screen test should be performed in order to define the appropriate dose level to test in the LLNA. The purpose of the pre-screen test is to provide guidance for selecting the maximum dose level to use in the main LLNA study, where information on the concentration that induces systemic toxicity (see paragraph 24) and/or excessive local skin irritation (see paragraph 23) is not available. The maximum dose level tested should be 100 % of the test substance for liquids or the maximum possible concentration for solids or suspensions.

• 22.The pre-screen test is conducted under conditions identical to the main LLNA study, except there is no assessment of lymph node proliferation and fewer animals per dose group can be used. One or two animals per dose group are suggested. All mice will be observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Body weights are recorded pre-test and prior to termination (Day 6). Both ears of each mouse are observed for erythema and scored using Table 1 (25). Ear thickness measurements are taken using a thickness gauge (e.g. digital micrometer or Peacock Dial thickness gauge) on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose), and Day 6. Additionally, on Day 6, ear thickness could be determined by ear punch weight determinations, which should be performed after the animals are humanely killed. Excessive local skin irritation is indicated by an erythema score ≥ 3 and/or an increase in ear thickness of ≥ 25 % on any day of measurement (26) (27). The highest dose selected for the main LLNA study will be the next lower dose in the pre-screen concentration series (see paragraph 18) that does not induce systemic toxicity and/or excessive local skin irritation.

Table 1

Erythema Scores

Observation	Score
No erythema	0
Very slight erythema (barely perceptible)	1
Well-defined erythema	2
Moderate to severe erythema	3
Severe erythema (beet redness) to eschar formation preventing grading of erythema	4

• 23.In addition to a 25 % increase in ear thickness (26) (27), a statistically significant increase in ear thickness in the treated mice compared to control mice has also been used to identify irritants in the LLNA (28) (29) (30) (31) (32) (33) (34). However, while statistically significant increases can occur when ear thickness is less than 25 % they have not been associated specifically with excessive irritation (30) (32) (33) (34).

• 24.The following clinical observations may indicate systemic toxicity (35) (36) when used as part of an integrated assessment and therefore may indicate the maximum dose level to use in the main LLNA: changes in nervous system function (e.g. pilo-erection, ataxia, tremors, and convulsions); changes in behaviour (e.g. aggressiveness, change in grooming activity, marked change in activity level); changes in respiratory patterns (i.e. changes in frequency and intensity of breathing such as dyspnea, gasping, and rales), and changes in food and water consumption. In addition, signs of lethargy and/or unresponsiveness and any clinical signs of more than slight or momentary pain and distress, or a > 5 % reduction in body weight from Day 1 to Day 6, and mortality should be considered in the evaluation. Moribund animals or animals obviously in pain or showing signs of severe and enduring distress should be humanely killed (37).

Main study experimental schedule

• 25.The experimental schedule of the assay is as follows:

• — Day 1 : Individually identify and record the weight of each animal and any clinical observation. Apply 25 μL of the appropriate dilution of the test substance, the vehicle alone, or the PC (concurrent or recent, based on laboratory policy in considering paragraphs 11-15), to the dorsum of each ear.

• — Days 2 and 3 : Repeat the application procedure carried out on Day 1.

• — Days 4 and 5 : No treatment.