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)

The neuropathological examination should be designed to complement and extend the observations made during the in vivo phase of the study. Tissues from at least five animals/sex/group (see Table 1 and next paragraph) should be fixed in situ, using generally recognised perfusion and fixation techniques (see reference 3, chapter 5 and reference 4, chapter 50). Any observable gross changes should be recorded. When the study is conducted as a stand-alone study screen for neurotoxicity or to characterise neurotoxic effects, the remainder of the animals may be used either for specific neurobehavioural (10)(11), neuropathological (10)(11)(12)(13), neurochemical (10)(11)(14)(15) or electrophysiological (10)(11)(16)(17) procedures that may supplement the procedures and examinations described here, or to increase the number of subjects examined for histophatology. These supplementary procedures are of particular use when empirical observations or anticipated effects indicate a specific type or target of neurotoxicity (2)(3). Alternatively, the remainder of the animals can also be used for routine pathological evaluations as described in Method for repeated dose studies.

A general staining procedure, such as haematoxylin and eosin (H&E), should be performed on all tissue specimens embedded in paraffin and microscopic examination should be carried out. If signs of peripheral neuropathy are observed or suspected, plastic-embedded samples of peripheral nerve tissue should be examined. Clinical signs may also suggest additional sites for examination or the use of special staining procedures. Guidance on additional sites to be examined can be found in (3)(4). Appropriate special stains to demonstrate specific types of pathological change may also be helpful (18).

Representative sections of the central and peripheral nervous system should be examined histologically (see reference 3, chapter 5 and reference 4, chapter 50). The areas examined should normally include: the forebrain, the centre of the cerebrum, including a section through the hippocampus, the midbrain, the cerebellum, the pons, the medulla oblongata, the eye with optic nerve and retina, the spinal cord at the cervical and lumbar swellings, the dorsal root ganglia, the dorsal and ventral root fibres, the proximal sciatic nerve, the proximal tibial nerve (at the knee) and the tibial nerve calf muscle branches. The spinal cord and peripheral nerve sections should include both cross or transverse and longitudinal sections. Attention should be given to the vasculature of the nervous system. A sample of skeletal muscle, particularly calf muscle, should also be examined. Special attention should be paid to sites with cellular and fibre structure and pattern in the CNS and PNS known to be particularly affected by neurotoxicants.

Guidance on neurophatological alterations that typically result from toxicant exposure can be found in the references (3)(4). A stepwise examination of tissue samples is recommended in which sections from the high dose group are first compared with those of the control group. If no neurophatological alterations are observed in the samples from these groups, subsequent analysis is not required. If neuropathological alterations are observed in the high dose group, sample from each of the potentially affected tissues from the intermediate and low dose groups should then be coded and examined sequentially.

If any evidence of neuropathological alterations is found in the qualitative examination, then a second examination should be performed on all regions of the nervous system showing these alterations. Sections from all dose groups from each of the potentially affected regions should be coded and examined at random without knowledge of the code. The frequency and severity of each lesion should be recorded. After all regions from all dose groups have been rated, the code can be broken and statistical analysis performed to evaluate dose-response relationships. Examples of different degrees of severity of each lesion should be described.

The neuropathological findings should be evaluated in the context of behavioural observations and measurements, as well as other data from preceding and concurrent systemic toxicity studies of the test substance.

2. DATA

2.1.   TREATMENT OF RESULTS

Individual data should be provided. Additionally, all data should be summarised in tabular form showing for each test or control group the number of animals at the start of the test, the number of animals found dead during the test or killed for humane reasons and the time of any death or humane kill, the number showing signs of toxicity, a description of the signs of toxicity observed, including time of onset, duration, type and severity of any toxic effects, the number of animals showing lesions, including the type and severity of the lesion(s).

2.2.   EVALUATION AND INTERPRETATION OF RESULTS

The findings of the study should be evaluated in terms of the incidence, severity and correlation of neurobehavioural and neuropathological effects (neurochemical or electrophysiological effects as well if supplementary examinations are included) and any other adverse effects observed. When possible, numerical results should be evaluated by an appropriate and generally acceptable statistical method. The statistical methods should be selected during the design of the study.

3. REPORTING

3.1.   TEST REPORT

the test report must include the following information:

Test substance:

• — physical nature (including isomerism, purity and physicochemical properties),

• — identification data.

Vehicle (if appropriate):

• — justification for choice of vehicle.

Test animals:

• — species/strain used,

• — number, age and sex of animals,

• — source, housing conditions, acclimatisation, diet, etc,

• — individual weights of animals at the start of the test.

Test conditions:

• — details of test substance formulation/diet preparation, achieved concentration, stability and homogeneity of the preparation,

• — specification of the doses administered, including details of the vehicle, volume and physical form of the material administered,

• — details of the administration of the test substance,

• — rationale for dose levels selected,

• — rationale for the route and duration of the exposure,

• — conversion from diet/drinking water test substance concentration (ppm) to the actual dose (mg/kg body weight/day), if applicable,

• — details of the food and water quality.

Observation and test procedures:

• — details of the assignment of animals in each group to the perfusion subgroups,

• — details of scoring systems, including criteria and scoring scales for each measurement in the detailed clinical observations,

• — details on the functional tests for sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive), for assessment of limb grip strength, for motor activity assessment (including details of automated devices for detecting activity), and other procedures used,

• — details of ophthalmologic examinations and, if appropriate, haematological examinations and clinical biochemistry tests with relevant base-line values,

• — details for specific neurobehavioural, neuropathological, neurochemical or electrophysiological procedures.

Results:

• — body weight/body weight changes including body weight at kill,

• — food consumption and water consumption, as appropriate,

• — toxic response data by sex and dose level, including signs of toxicity or mortality,

• — nature, severity and duration (time of onset and subsequent course) of the detailed clinical observations (whether reversible or not),

• — a detailed description of all functional test results,

• — necropsy findings,

• — a detailed description of all neurobehavioural, neuropathological, and neurochemical or electrophysiological findings, if available,

• — absorption and metabolism data, if available,

• — statistical treatment of results, where appropriate.

Discussion of results:

• — dose response information;

• — relationship of any other toxic effects to a conclusion about the neurotoxic potential of the test chemical;

• — no-observed-adverse effect level.

Conclusions:

• — a specific statement of the overall neurotoxicity of the test chemical is encouraged.

4. REFERENCES

• (1)OECD Giudance Document on Neurotoxicity Testing Strategies and Test Methods. OECD, Paris, In Preparation.

• (2)Test Guideline for a Developmental Neurotoxicity Study, OECD Guidelines for the Testing of Chemicals. In preparation.

• (3)World Health Organisation (WHO) (1986) Environmental Health Criteria document 60: Principles and Methods for the Assessment of Neurotoxicity associated with Exposure to Chemicals.

• (4)Spencer, P.S. and Schaumburg, H.H. (1980) Experimental and Clinical Neurotoxicology. Eds. Spencer, P.S. and Schaumburg, H.H. eds. Williams and Wilkins, Baltimore/London.

• (5)Tupper, D.E. and Wallace, R.B., (1980) Utility of the Neurological Examination in Rats. Acta Neurobiol. Exp., 40, p. 999-1003.

• (6)Gad, S.C., (1982) A Neuromuscular Screen for Use in Industrial Toxicology. J. Toxicol. Environ. Health, 9, p. 691-704.

• (7)Moser, V.C., McDaniel, K.M. and Phillips, P.M., (1991) Rat Strain and Stock Comparisons Using a Functional Observational Battery: Baseline Values and Effects of amitraz. Toxic. Appl. Pharmacol., 108, p. 267-283.

• (8)Meyer, O.A., Tilson, H.A., Byrd, W.C. and Riley, M.T., (1979) A Method for the Routine Assessment of Fore- and Hind- limb Grip Strength of Rats and Mice. Neurobehav. Toxicol., 1, p. 233-236.

• (9)Crofton, K.M., Haward, J.L., Moser, V.C., Gill, M.W., Reirer, L.W., Tilson, H.A. and MacPhail, R.C. (1991) Interlaboratory Comparison of Motor Activity Experiments: Implication for Neurotoxicological Assessments. Neurotoxicol. Teratol., 13, p. 599-609.

• (10)Tilson, H.A., and Mitchell, C.L. eds., (1992) Neurotoxicology Target Organ Toxicology Series. Raven Press, New York.

• (11)Chang, L.W., ed., (1995) Principles of Neurotoxicology. Marcel Dekker, New York.

• (12)Broxup, B., (1991) Neuopathology as a screen for Neurotoxicity Assessment. J. Amer. Coll. Toxicol., 10, p. 689-695.

• (13)Moser, V.C., Anthony, D.C., Sette, W.F. and MacPhail, R.C., (1992) Comparison of Subchronic Neurotoxicity of 2-Hydroxyethyl Acrylate and Acrylamide in Rats. Fund. Appl.Toxicol., 18, p. 343-352.

• (14)O'Callaghan, J.P. (1988). Neurotypic and Gliotypic Proteins as Biochemical Markers of Neurotoxicity. Eurotoxicol. Teratol., 10, p. 445-452.

• (15)O'Callaghan J.P. and Miller, D.B., (1988) Acute Exposure of the Neonatal Rat to Triethyltin Results in Persistent Changes in Neurotypic and Gliotypic Proteins. J. Pharmacol. Exp. Ther., 244, p. 368-378.

• (16)Fox. D.A., Lowndes, H.E. and Birkamper, G.G., (1982) Electrophysiological Techniques in Neurotoxicology. In: Nervous System Toxicology. Mitchell, C.L. ed. Raven Press, New York, p. 299-335.

• (17)Johnson, B.L., (1980) Electrophysiological Methods in neurotoxicity Testing. In: Experimental and Clinical Neurotoxicology. Spencer, P.S. and Schaumburg, H.H. eds., Williams and Wilkins Co.,. Baltimore/London, p. 726-742.

• (18)Bancroft, J.D. and Steven A., (1990) Theory and Pratice of Histological Techniques. Chapter 17, Neuropathological Techniques. Lowe, James and Cox, Gordon eds. Churchill Livingstone.

Table 1

Minimum numbers of animals needed per group when the neurotoxicity study is conducted separately or in combination whit studies

	NEUROTOXICITY STUDY CONDUCTED AS:	NEUROTOXICITY STUDY CONDUCTED AS:	NEUROTOXICITY STUDY CONDUCTED AS:	NEUROTOXICITY STUDY CONDUCTED AS:
	Separate study	Combined study with the 28-day study	Combined study with the 90-day study	Combined study with the chronic toxicity study
Total number of animals per group	10 males and 10 females	10 males and 10 females	15 males and 15 females	25 males and 25 females
Number of animals selected for functional testing including detailed clinical observations	10 males and 10 females	10 males and 10 females	10 males and 10 females	10 males and 10 females
Number of animals selected per perfusion in situ and neurohistopathology	5 males and 5 females	5 males and 5 females	5 males and 5 females	5 males and 5 females
Number of animals selected for repeated dose/subchronic/chronic toxicity observations, haematology, clinical biochemistry, histopathology, etc. as indicate in the respective Guidelines		5 males and 5 females	10 males [^E0029] and 10 females [^E0029]	20 males [^E0029] and 20 females [^E0029]
Supplemental observations, as appropriate	5 males and 5 females

†  Includes five animals selected for functional testing and detailed clinical observations as part of the neurotoxicity study.

Table 2

Frequency of clinical observation and functional tests

Type of observations	Type of observations	Study duration	Study duration	Study duration	Study duration
Type of observations	Type of observations	Acute	28-day	90-day	Chronic
In all animals	General health condition	daily	daily	daily	daily
In all animals	Mortality/morbidity	Twice daily	Twice daily	Twice daily	Twice daily
In animals selected for functional observations	Detailed clinical observations	— prior to first exposure — within 8 hours of dosing at estimate time of peak effect — at day 7 and 14 after dosing	— prior to first exposure — once weekly thereafter	— prior to first exposure — once during the first or second week of exposure — monthly thereafter	— prior to first exposure — once at the end of the first month of exposure — every three months thereafter
In animals selected for functional observations	Functional tests	— prior to first exposure — within 8 hours of dosing at estimate time of peak effect — at day 7 and 14 after dosing	— prior to first exposure — during the fourth week of treatment as close as possible to the end of the exposure period	— prior to first exposure — once during the first or second week of exposure — monthly thereafter	— prior to first exposure — once at the end of the first month of exposure — every three months thereafter

B.44.   SKIN ABSORPTION: IN VIVO METHOD

1. METHOD

This testing method is equivalent to the OECD TG 427 (2004).

1.1.   INTRODUCTION

Exposure to many chemicals occurs mainly via the skin whilst the majority of toxicological studies performed in laboratory animals use the oral route of administration. The in vivo percutaneous absorption study set out in this guideline provides the linkage necessary to extrapolate from oral studies when making safety assessments following dermal exposure.

A substance must cross a large number of cell layers of the skin before it can reach the circulation. The rate-determining layer for most substances is the stratum corneum consisting of dead cells. Permeability through the skin depends both on the lipophilicity of the chemical and the thickness of the outer layer of epidermis, as well on factors such as molecular weight and concentration of the substance. In general, the skin of rats and rabbits is more permeable than that of humans, whereas the skin permeability of guinea pigs and monkeys is more similar to that of humans.

The methods for measuring percutaneous absorption can be divided into two categories; in vivo and in vitro.The in vivo method is capable of providing good information, in various laboratory species, on skin absorption. More recently in vitro methods have been developed. These utilise transport across full or partial thickness animal or human skin to a fluid reservoir. The in vitro method is described in a separate testing method (1). It is recommended that the OECD Guidance Document for the Conduct of Skin Absorption Studies (2) be consulted to assist in the selection of the most appropriate method in the given situation, as it provides more details on the suitability of both in vivo and in vitro methods.

The in vivo method, described in this method, allows the determination of the penetration of the test substance through the skin into the systemic compartment. The technique has been widely used for many years (3)(4)(5)(6)(7). Although in vitro percutaneous absorption studies may in many cases be appropriate there may be situations in which only an in vivo study can provide the necessary data.

Advantages of the in vivo method are that it uses a physiologically and metabolically intact system, uses a species common to many toxicity studies and can be modified for use with other species. The disadvantages are the use of live animals, the need for radiolabelled material to facilitate reliable results, difficulties in determining the early absorption phase and the differences in permeability of the preferred species (rat) and human skin. Animal skin is generally more permeable and therefore may overestimate human percutaneous absorption (6)(8)(9). Caustic/corrosive substances should not be tested in live animals.

1.2.   DEFINITIONS

Unabsorbed dose: represents that washed from the skin surface after exposure and any present on the non-occlusive cover, including any dose shown to volatilise from the skin during exposure.

Absorbed dose (in vivo): comprises that present in urine, cage wash, faeces, expired air (if measured), blood, tissues (if collected) and the remaining carcass, following removal of application site skin.

Absorbable dose: represents that present on or in the skin following washing.

1.3.   PRINCIPLE OF THE TEST METHOD

The test substance, preferably radiolabelled, is applied to the clipped skin of animals at one or more appropriate dose levels in the form of a representative in-use preparation. The test preparation is allowed to remain in contact with the skin for a fixed period of time under a suitable cover (non-occlusive, semi-occlusive, or occlusive) to prevent ingestion of the test preparation. At the end of the exposure time the cover is removed and the skin is cleaned with an appropriate cleansing agent, the cover and the cleansing materials are retained for analysis and a fresh cover applied. The animals are housed prior to, during and after the exposure period in individual metabolism cages and the excreta and expired air over these periods are collected for analysis. The collection of expired air can be omitted when there is sufficient information that little or no volatile radioactive metabolite is formed. Each study will normally involve several groups of animals that will be exposed to the test preparation. One group will be killed at the end of the exposure period. Other groups will be killed at scheduled time intervals thereafter (2). At the end of the sampling time the remaining animals are killed, blood is collected for analysis, the application site removed for analysis and the carcass is analysed for any unexcreted material. The samples are assayed by appropriate means and the degree of percutaneous absorption is estimated (6)(8)(9).

1.4.   DESCRIPTION OF THE METHOD

1.4.1.   Selection of animal species

The rat is the most commonly used species, but hairless strains and species having skin absorption rates more similar to those of human, can also be used (3)(6)(7)(8)(9). Young adult healthy animals of a single sex (with males as the default sex) of commonly used laboratory strains should be employed. At the commencement of the study, the weight variation of animals used should not exceed ± 20 % of the mean weight. As an example, male rats of 200 g – 250 g are suitable, particularly in the upper half of this range.

1.4.2.   Number and sex of animals

A group of at least four animals of one sex should be used for each test preparation and each scheduled termination time. Each group of animals will be killed after different time intervals, for example at the end of the exposure period (typically 6 or 24 hours) and subsequent occasions (e.g. 48 and 72 hours). If there are data available that demonstrate substantial differences in dermal toxicity between males and females, the more sensitive sex should be chosen. If there are no such data, then either gender can be used.

1.4.3.   Housing and feeding conditions

The temperature in 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 and should be freely available together with an unlimited supply of drinking water. During the study, and preferably also during the acclimatisation, the animals are individually housed in metabolism cages. Since food and water spillage would compromise the results, the probability of such events should be minimised.

1.4.4.   Preparation of animals

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

Following the acclimatisation period, and approximately 24 hours prior to dosing, each animal will have an area of skin in the region of the shoulders and the back clipped. The permeation properties of damaged skin are different from intact skin and care should be taken to avoid abrading the skin. Following the clipping and approximately 24 hours before the test substance is applied to the skin, (See Section 1.4.7) the skin surface should be wiped with acetone to remove sebum. An additional soap and water wash is not recommended because any soap residue might promote test substance absorption. The area must be large enough to allow reliable calculation of the absorbed amount of test chemical per cm² skin, preferably at least 10 cm². This area is practicable with rats of 200-250 g bodyweight. After preparation, the animals are returned to metabolism cages.

1.4.5.   Test substance

The test substance is the entity whose penetration characteristics are to be studied. Ideally, the test substance should be radiolabelled.

1.4.6.   Test preparation

The test substance preparation (e.g. neat, diluted, or formulated material containing the test chemical which is applied to the skin) should be the same (or realistic surrogate) as that to which humans or other potential target species may be exposed. Any variations from the ‘in-use’ preparation must be justified. Where necessary, the test substance is dissolved or suspended in a suitable vehicle. For vehicles other than water the absorption characteristics and potential interaction with the test substance should be known.

1.4.7.   Application to the skin

An application site of a specific surface area is defined on the skin surface. A known amount of the test preparation is then evenly applied to the site. This amount should normally mimic potential human exposure, typically 1-5 mg/cm² for a solid or up to 10 μl/cm² for liquids. Any other quantities should be justified by the expected use conditions, the study objectives or physical characteristics of the test preparation. Following application, the treated site must be protected from grooming. An example of a typical device is shown in Figure 1. Normally, the application site will be protected by a non-occlusive cover (e.g. a permeable nylon gauze cover). However, for infinite applications the application site should be occluded. In case of evaporation of semivolatile test substances reduces the recovery rate of the test substance to an unacceptable extend (see also section 1.4.10, first paragraph), it is necessary to trap the evaporated substance in a charcoal filter covering the application device (see Figure 1). It is important that any device does not damage the skin, nor absorb or react with the test preparation. The animals are returned to individual metabolism cages in order to collect excreta.

1.4.8.   Duration of exposure and sampling

The duration of exposure is the time interval between application and removal of test preparation by skin washing. A relevant exposure period (typically 6 or 24 hours) should be used, based on the expected human exposure duration. Following the exposure period, the animals are maintained in the metabolism cages until the scheduled termination. The animals should be observed for signs of toxicity/abnormal reactions at regular intervals for the entire duration of the study. At the end of the exposure period the treated skin should be observed for visible signs of irritation.

The metabolism cages should permit separate collection of urine and faeces throughout the study. They should also allow collection of ¹⁴C-carbon dioxide and volatile ¹⁴C-carbon compounds, which should be analysed when produced in quantity (> 5 %). The urine, faeces and trap fluids (e.g. ¹⁴C-carbon dioxide and volatile ¹⁴C-compounds) should be individually collected from each group at each sampling time. If there is sufficient information that little or no volatile radioactive metabolite is formed, open cages can be used.

Excreta are collected during the exposure period, up to 24 hours after the initial skin contact and then daily until the end of the experiment. Whilst three excreta collection intervals will normally be sufficient, the envisaged purpose of the test preparation or existing kinetic data may suggest more appropriate or additional time points for study.

At the end of the exposure period the protective device is removed from each animal and retained separately for analysis. The treated skin of all animals should be washed at least three times with cleansing agent using suitable swabs. Care must be taken to avoid contaminating other parts of the body. The cleansing agent should be representative of normal hygiene practice, e.g. aqueous soap solution. Finally, the skin should be dried. All swabs and washings must be retained for analysis. A fresh cover should be applied to protect the treated site of those animals forming later time point groups prior to their return to individual cages.

1.4.9.   Terminal procedures

For each group, the individual animals should be killed at the scheduled time and blood collected for analysis. The protective device or cover should be removed for analysis. The skin from the application site and a similar area of non-dosed, clipped skin should be removed from each animal for separate analysis. The application site may be fractioned to separate the stratum corneum from the underlying epidermis to provide more information on the test chemical disposition. The determination of this disposition over a time course after the exposure period should provide some indication of the fate of any test chemical in the stratum corneum. To facilitate skin fractionation (following the final skin wash and killing the animal) each protective cover is removed. The application site skin, with annular ring of surrounding skin, is excised from the rat and pinned on a board. A strip of adhesive tape is applied to the skin surface using gentle pressure and the tape removed together with part of the stratum corneum. Successive strips of tape are applied until the tape no longer adheres to the skin surface, when all of the stratum corneum has been removed. For each animal, all the tape strips may be combined in a single container to which a tissue digestant is added to solubilise the stratum corneum. Any potential target tissues may be removed for separate measurement before the residual carcass is analysed for absorbed carcass dose. The carcasses of the individual animals should be retained for analysis. Usually analysis of the total content will be sufficient. Target organs may be removed for separate analysis (if indicated by other studies). Urine present in the bladder at scheduled kill should be added to the previous urine collection. After collection of the excreta from metabolism cages at the time scheduled kill, the cages and their traps should be washed with an appropriate solvent. Other potentially contaminated equipment should likewise be analysed.

1.4.10.   Analysis

In all studies adequate recovery (i.e. mean of 100 ± 10 % of the radioactivity) should be achieved. Recoveries outside this range must be justified. The amount of the administered dose in each sample should be analysed by suitably validated procedures.

Statistical considerations should include a measure of variance for the replicates for each application.

2. DATA

The following measurements should be made for each animal, at each sampling time for the test chemical and/or metabolites. In addition to individual data, data grouped according to sampling times should be reported as means.

• — quantity associated with the protective appliances,

• — quantity that can be dislodged from the skin,

• — quantity in/on skin that cannot be washed from the skin,

• — quantity in the sampled blood,

• — quantity in the excreta and expired air (if appropriate),

• — quantity remaining in the carcass and any organs removed for separate analysis.

The quantity of test substance and/or metabolites in the excreta, expired air, blood and in the carcass will allow determination of the total amount absorbed at each time point. A calculation of the amount of test chemical absorbed per cm² of skin exposed to the test substance over the exposure period can also be obtained.

3. REPORTING

3.1.   TEST REPORT

The test report must include the requirements stipulated in the protocol, including a justification for the test system used and should comprise the following:

test substance:

• — identification data (e.g. CAS number, if available, source, purity (radiochemical purity), known impurities, lot number),

• — physical nature, physicochemical properties (e.g. pH, volatility, solubility, stability, molecular weight and log Pow).

Test preparation:

• — formulation and justification of use,

• — details of the test preparation, amount applied, achieved concentration, vehicle, stability and homogeneity.

Test animal:

• — species/strain used,

• — number, age and sex of animals,

• — source of animals, housing conditions, diets, etc.,

• — individual animal weights at start of test.

Test conditions:

• — details of the administration of the test preparation (site of application, assay methods, occlusion/non-occlusion, volume, extraction, detection),

• — details of food and water quality.

Results:

• — any signs of toxicity,

• — tabulated absorption data (expressed as rate, amount or percentage),

• — overall recoveries of the experiment,

• — interpretation of the results, comparison with any available data on percutaneous absorption of the test compound.

Discussion of the results.

Conclusions.

4. REFERENCES

• (1)Testing Method B.45. Skin Absorption: In vitro Method.

• (2)OECD (2002). Guidance Document for the Conduct of Skin Absorption Studies. OECD, Paris.

• (3)ECETOC, (1993) Percutaneous Absorption. European Centre for Ecotoxicology and Toxicology of Chemicals, Monograph No 20.

• (4)Zendzian R.P. (1989) Skin Penetration Method suggested for Environmental Protection Agency Requirements. J. Am. Coll. Toxicol. 8(5), p. 829-835.

• (5)Kemppainen, B.W., Reifenrath WG (1990) Methods for skin absorption. CRC Press Boca Raton, FL, USA.

• (6)EPA, (1992) Dermal Exposure Assessment: Principles and Applications. Exposure Assessment Group, Office of Health and Environmental Assessment.

• (7)EPA, (1998) Health Effects Test Guidelines, OPPTS 870-7600, Dermal Penetration. Office of Prevention, Pedsticides and Toxic Substances.

• (8)Bronaugh, R.L., Wester, R.C., Bucks, D., Maibach H.I. and Sarason, R., (1990) In vivo percutaneous absorption of fragrance ingredients in reshus monkeys and humans. Fd. Chem. Toxic. 28, p. 369-373.

• (9)Feldman, R.J. and Maibach, H.I., (1970) Absorption of some organic compounds through the skin in man. J. Invest Dermatol. 54, p. 399-404.

Figure 1

An example of a design of a typical device used to define and protect dermal application site during in vivo percutaneous absorption studies

Image omitted; see official source

Text of image

Activated charcoal filter or gauze cover

Screw-threaded cap

Screw-threaded base

Cyanoacrylate glue

Skin

B.45.   SKIN ABSORPTION: IN VITRO METHOD

1. METHOD

This testing method is equivalent to the OECD TG 428 (2004).

1.1.   INTRODUCTION

This method has been designed to provide information on absorption of a test substance applied to excised skin. It can either be combined with the method for skin absorption: in vivo method (1), or be conducted separately. It is recommended that the OECD Guidance Document for the Conduct of Skin Absorption Studies (2) be consulted to assist in the design of studies based on this method. The Guidance Document has been prepared to facilitate the selection of appropriate in vitro procedures for use in specific circumstances, to ensure the reliability of results obtained by this method.

The methods for measuring skin absorption and dermal delivery can be divided into two categories: in vivo and in vitro. In vivo methods on skin absorption are well established and provide pharmacokinetic information in a range of animal species. An in vivo method is separately described in another testing method (1). In vitro methods have also been used for many years to measure skin absorption. Although formal validation studies of the in vitro methods covered by this testing method have not been performed, OECD experts agreed in 1999 that there was sufficient data evaluated to support the in vitro method (3). Further details that substantiate this support, including a significant number of direct comparisons of in vitro and in vivo methods, are provided with the Guidance Document (2). There are a number of monographs that review this topic and provide detailed background on the use of an in vitro method (4)(5)(6)(7)(8)(9)(10)(11)(12). In vitro methods measure the diffusion of chemicals into and across skin to a fluid reservoir and can utilise non-viable skin to measure diffusion only, or fresh, metabolically active skin to simultaneously measure diffusion and skin metabolism. Such methods have found particular use as a screen for comparing delivery of chemicals into and through skin from different formulations and can also provide useful models for the assessment of percutaneous absorption in humans.

The in vitro method may not be applicable for all situations and classes of chemicals. It may be possible to use the in vitro test method for an initial qualitative evaluation of skin penetration. In certain cases, it may be necessary to follow this up with in vivo data. The guidance document (2) should be consulted for further elaboration of situations where the in vitro method would be suitable. Additional detailed information to support the decision is provided in (3).

This method presents general principles for measuring dermal absorption and delivery of a test substance using excised skin. Skin from many mammalian species, including humans, can be used. The permeability properties of skin are maintained after excision from the body because the principal diffusion barrier is the non-viable stratum corneum; active transport of chemicals through the skin has not been identified. The skin has been shown to have the capability to metabolise some chemicals during percutaneous absorption (6), but this process is not rate limiting in terms of actual absorbed dose, although it may affect the nature of the material entering the bloodstream.

1.2.   DEFINITIONS

Unabsorbed dose: represents that washed from the skin surface after exposure and any present on the non-occlusive cover, including any dose shown to volatilise from the skin during exposure.

Absorbed dose (in vitro): mass of test substance reaching the receptor fluid or systemic circulation within a specified period of time.

Absorbable dose (in vitro): represents that present on or in the skin following washing.

1.3.   PRINCIPLE OF THE TEST METHOD

The test substance, which may be radiolabelled, is applied to the surface of a skin sample separating the two chambers of a diffusion cell. The chemical remains on the skin for a specified time under specified conditions, before removal by an appropriate cleansing procedure. The receptor fluid is sampled at time points throughout the experiment and analysed for the test chemical and/or metabolites.

When metabolically active systems are used, metabolites of the test chemical may be analysed by appropriate methods. At the end of the experiment the distribution of the test chemical and its metabolites are quantified, when appropriate.

Using appropriate conditions, which are described in this method and the guidance document (2), absorption of a test substance during a given time period is measured by analysis of the receptor fluid and the treated skin. The test substance remaining in the skin should be considered as absorbed unless it can be demonstrated that absorption can be determined from receptor fluid values alone. Analysis of the other components (material washed off the skin and remaining within the skin layers) allows for further data evaluation, including total test substance disposition and percentage recovery.

To demonstrate the performance and reliability of the test system in the performing laboratory, the results for relevant reference chemicals should be available and in agreement with published literature for the method used. This requirement could be met by testing an appropriate reference substance (preferably of a lipophilicity close to the test substance) concurrently with the test substance or by providing adequate historical data for a number of reference substances of different lipophilicity (e.g. caffeine, benzoic acid, and testosterone).

1.4.   DESCRIPTION OF THE TEST METHOD

1.4.1.   Diffusion cell

A diffusion cell consists of a donor chamber and a receptor chamber between which the skin is positioned (an example of a typical design is provided in Figure 1). The cell should provide a good seal around the skin, enable easy sampling and good mixing of the receptor solution in contact with the underside of the skin, and good temperature control of the cell and its contents. Static and flow-through diffusion cells are both acceptable. Normally, donor chambers are left unoccluded during exposure to a finite dose of a test preparation. However, for infinite applications and certain scenarios for finite doses, the donor chambers may be occluded.

1.4.2.   Receptor fluid

The use of a physiologically conducive receptor fluid is preferred although others may also be used provided that they are justified. The precise composition of the receptor fluid should be provided. Adequate solubility of the test chemical in the receptor fluid should be demonstrated so that it does not act as a barrier to absorption. In addition, the receptor fluid should not affect skin preparation integrity. In a flow-through system, the rate of flow must not hinder diffusion of a test substance into the receptor fluid. In a static cell system, the fluid should be continuously stirred and sampled regularly. If metabolism is being studied, the receptor fluid must support skin viability throughout the experiment.

1.4.3.   Skin preparations

Skin from human or animal sources can be used. It is recognised that the use of human skin is subject to national and international ethical considerations and conditions. Although viable skin is preferred, non-viable skin can also be used provided that the integrity of the skin can be demonstrated. Either epidermal membranes (enzymically, heat or chemically separated) or split thickness skin (typically 200-400 μm thick) prepared with a dermatome, are acceptable. Full thickness skin may be used but excessive thickness (approximately > 1 mm) should be avoided unless specifically required for determination of the test chemical in layers of the skin. The selection of species, anatomical site and preparative technique must be justified. Acceptable data from a minimum of four replicates per test preparation are required.

1.4.4.   Skin preparation integrity

It is essential that the skin is properly prepared. Inappropriate handling may result in damage to the stratum corneum, hence the integrity of the prepared skin must be checked. When skin metabolism is being investigated, freshly excised skin should be used as soon as possible, and under conditions known to support metabolic activity. As a general guidance, freshly excised skin should be used within 24 hours, but the acceptable storage period may vary depending on the enzyme system involved in metabolisation and storage temperatures (13). When skin preparations have been stored prior to use, evidence should be presented to show that barrier function is maintained.

1.4.5.   Test substance

The test substance is the entity whose penetration characteristics are to be studied. Ideally, the test substance should be radiolabelled.

1.4.6.   Test preparation

The test substance preparation (e.g. neat, diluted or formulated material containing the test substance which is applied to the skin) should be the same (or a realistic surrogate) as that to which humans or other potential target species may be exposed. Any variation from the ‘in-use’ preparation must be justified.

1.4.7.   Test substances concentrations and formulations

Normally more than one concentration of the test substance is used spanning the upper of potential human exposures. Likewise, testing a range of typical formulations should be considered.

1.4.8.   Application to the skin

Under normal conditions of human exposure to chemicals, finite doses are usually encountered. Therefore, an application that mimics human exposure, normally 1-5 mg/cm² of skin for a solid and up to10 μl/cm² for liquids, should be used. The quantity should be justified by the expected use conditions, the study objectives or physical characteristics of the test preparation. For example, applications to the skin surface may be infinite, where large volumes per unit area are applied.

1.4.9.   Temperature

The passive diffusion of chemicals (and therefore their skin absorption) is affected by temperature. The diffusion chamber and skin should be maintained at a constant temperature close to normal skin temperature of 32 ± 1 ^oC. Different cell designs will require different water bath or heated block temperatures to ensure that the receptor/skin is at its physiological norm. Humidity should preferably be between 30 and 70 %.

1.4.10.   Duration of exposure and sampling

Skin exposure to the test preparation may be for the entire duration of the experiment or for shorter times (i.e., to mimic a specific type of human exposure). The skin should be washed of excess test preparation with a relevant cleansing agent, and the rinses collected for analysis. The removal procedure of the test preparation will depend on the expected use condition, and should be justified. A period of sampling of 24 hours is normally required to allow for adequate characterisation of the absorption profile. Since skin integrity may start to deteriorate beyond 24 hours, sampling times should not normally exceed 24 hours. For test substances that penetrate the skin rapidly this may not be necessary but, for test substances that penetrate slowly, longer times may be required. Sampling frequency of the receptor fluid should allow the absorption profile of the test substance to be presented graphically.

1.4.11.   Terminal procedures

All components of the test system should be analysed and recovery is to be determined. This includes the donor chamber, the skin surface rinsing, the skin preparation and the receptor fluid/chamber. In some cases, the skin may be fractionated into the exposed area of skin and area of skin under the cell flange, and into stratum corneum, epidermis and dermis fractions, for separate analysis.

1.4.12.   Analysis

In all studies adequate recovery should be achieved (the aim should be a mean of 100±10 % of the radioactivity and any deviation should be justified). The amount of test substance in the receptor fluid, skin preparation, skin surface washings and apparatus rinse should be analysed, using a suitable t echnique.

2. DATA

The analysis of receptor fluid, the distribution of the test substance chemical in the test system and the absorption profile with time, should be presented. When finite dose conditions of exposure are used, the quantity washed from the skin, the quantity associated with the skin (and in the different skin layers if analysed) and the amount present in the receptor fluid (rate, and amount or percentage of applied dose) should be calculated. Skin absorption may sometimes be expressed using receptor fluid data alone. However, when the test substance remains in the skin at the end of the study, it may need to be included in the total amount absorbed (see paragraph 66 in reference (3)). When infinite dose conditions of exposure are used the data may permit the calculation of a permeability constant (Kp). Under the latter conditions, the percentage absorbed is not relevant.

3. REPORTING

3.1.   TEST REPORT

The test report must include the requirements stipulated in the protocol, including a justification for the test system used and should, comprise the following:

test substance:

• — physical nature, physicochemical properties (at least molecular weight and log Pow), purity (radiochemical purity),

• — identification information (e.g. batch number),

• — solubility in receptor fluid.

Test preparation:

• — formulation and justification of use,

• — homogeneity.

Test conditions:

• — sources and site of skin, method of preparation, storage conditions prior to use, any pre-treatment (cleaning, antibiotic treatments, etc.), skin integrity measurements, metabolic stat us, justification of use,

• — cell design, receptor fluid composition, receptor fluid flow rate or sampling times and procedures,

• — details of application of test preparation and quantification of dose applied,

• — duration of exposure,

• — details of removal of test preparation from the skin, e.g. skin rinsing,

• — details of analysis of skin and any fractionation techniques employed to demonstrate skin distribution,

• — cell and equipment washing procedures,

• — assay methods, extraction techniques, limits of detection and analytical method validation.

Results:

• — overall recoveries of the experiment (Applied dose ≡ Skin washings + Skin + Receptor fluid + Cell washings),

• — tabulation of individual cell recoveries in each compartment,

• — absorption profile,

• — tabulated absorption data (expressed as rate, amount or percentage).

Discussion of results.

Conclusions.

4. REFERENCES

• (1)Testing Method B.44. Skin Absorption: In Vivo Method.

• (2)OECD, (2002) Guidance Document for the Conduct of Skin Absorption Studies. OECD, Paris.

• (3)OECD, (2000) Report of the Meeting of the OECD Extended Steering Committee for Percutaneous Absorption Testing, Annex 1 to ENV/JM/TG(2000)5. OECD, Paris.

• (4)Kemppainen B.W. and Reifenrath W.G., (1990) Methods for skin absorption. CRC Press, Boca Raton.

• (5)Bronaugh R.L. and Collier, S.W., (1991) Protocol for In vitro Percutaneous Absorption Studies, in In vitro Percutaneous Absorption: Principles, Fundamentals and Applications, RL Bronaugh and HI Maibach, Eds., CRC Press, Boca Raton, p. 237-241.

• (6)Bronaugh R.L. and Maibach H.I., (1991) In vitro Percutaneous Absorption: Principles, Fundamentals and Applications. CRC Press, Boca Raton.

• (7)European Centre for Ecotoxicology and Toxicology of Chemicals, (1993) Monograph No 20, Percutaneous Absorption, ECETOC, Brussels.

• (8)Diembeck W, Beck H, Benech-Kieffer F, Courtellemont P, Dupuis J, Lovell W, Paye M, Spengler J, Steiling W., (1999) Test Guidelines for In Vitro Assessment of Dermal Absorption and Percutaneous Penetration of Cosmetic Ingredients, Fd Chem Tox, 37, p. 191-205.

• (9)Recommended Protocol for In vitro Percutaneous Absorption Rate Studies (1996). US Federal Register, Vol. 61, No 65.

• (10)Howes, D., Guy, R., Hadgraft, J., Heylings, J.R. et al. (1996). Methods for assessing Percutaneous absorption. ECVAM Workshop Report ATLA 24, 81 R10.

• (11)Schaefer, H. and Redelmeier, T.E., (1996). Skin barrier: principles of percutaneous absorption. Karger, Basel.

• (12)Roberts, M.S. and Walters, K.A., (1998). Dermal absorption and toxicity assessment. Marcel Dekker, New York.

• (13)Jewell, C., Heylings, J.R., Clowes, H.M. and Williams, F.M. (2000). Percutaneous absorption and metabolism of dinitrochlorobenzene in vitro. Arch Toxicol 74, p. 356-365.

Figure 1

An example of a typical design of a static diffusion cell for in vitro percutaneous absorption studies

Image omitted; see official source

Text of image

Activated charcoal filter (for volatile test substances)

Cell donor chamber

Skin membrane (2.54 cm²)

Support grid

Glass diffusion cell

Magnetic stirrer bar

Receptor chamber/ fluid maintained at 32°C ± 1°C in a temperature-controlled water bath

(AUTO) SAMPLER

Programmed to sample specific time-course

(Auto) pipette/syringe sampling into scintillation or HPLC vials

Volume maintained by replacement of fresh receptor fluid

B.46.   IN VITRO SKIN IRRITATION: RECONSTRUCTED HUMAN EPIDERMIS 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.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

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

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

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

B.49.   IN VITRO MAMMALIAN CELL MICRONUCLEUS TEST

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

B.50.   SKIN SENSITISATION: LOCAL LYMPH NODE ASSAY: DA

INTRODUCTION

• 1.OECD Guidelines for the Testing of Chemicals and EU Test Methods are periodically reviewed in light of scientific progress, changing regulatory needs, and animal welfare considerations. The first Test Method (TM) (B.42) for the determination of skin sensitisation in the mouse, the Local Lymph Node Assay (LLNA; OECD Test Guideline 429) has been revised (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). In the LLNA, radioisotopic thymidine or iodine is used to measure lymphocyte proliferation and therefore the assay has limited use where the acquisition, use, or disposal of radioactivity is problematic. The LLNA: DA (developed by Daicel Chemical Industries, Ltd) is a non-radioactive modification to the LLNA, which quantifies adenosine triphosphate (ATP) content via bio-luminescence as an indicator of lymphocyte proliferation. The LLNA: DA test method has been validated and reviewed and recommended by an international peer review panel as considered useful for identifying skin sensitising and non-sensitising chemicals, with certain limitations (10) (11) (12) (13). This TM is designed for assessing skin sensitisation potential of chemicals (substances and mixtures) in animals. Chapter B.6 of this Annex and OECD Test Guideline 406 utilise guinea pig tests, notably the guinea pig maximisation test and the Buehler test (14) The LLNA (Chapter B.42 of this Annex; OECD Test Guideline 429) and the two non-radioactive modifications, LLNA: DA (Chapter B.50 of this Annex; OECD Test Guideline 442 A) and LLNA: BrdU-ELISA (Chapter B.51 of this Annex; OECD Test Guideline 442 B), all provide an advantage over the guinea pig tests in B.6 and OECD Test Guideline 406 (14) in terms of reduction and refinement of animal use.

• 2.Similar to the LLNA, the LLNA: DA studies the induction phase of skin sensitisation and provides quantitative data suitable for dose-response assessment. Furthermore, an ability to detect skin sensitisers without the necessity for using a radiolabel for DNA eliminates the potential for occupational exposure to radioactivity and waste disposal issues. This in turn may allow for the increased use of mice to detect skin sensitisers, which could further reduce the use of guinea pigs to test for skin sensitisation potential (i.e. B.6; OECD Test Guideline 406) (14).

DEFINITIONS

• 3.Definitions used are provided in Appendix 1.

INITIAL CONSIDERATIONS AND LIMITATIONS

• 4.The LLNA: DA is a modified LLNA method for identifying potential skin sensitising chemicals, with specific limitations. This does not necessarily imply that in all instances the LLNA: DA should be used in place of the LLNA or guinea pig tests (i.e. B.6; OECD Test Guideline 406) (14), 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 (10) (11). 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: DA is appropriate for the test substance (given the incompatibility of limited types of chemicals with the LLNA: DA (see paragraph 5) and to aid in dose selection.

• 5.The LLNA: DA 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 animal use for this purpose when compared to the guinea pig tests (B.6; OECD Test Guideline 406) (14). Moreover, the LLNA: DA offers a substantial refinement (less pain and distress) of the way in which animals are used for allergic contact sensitisation testing, since unlike the B.6 and OECD Test Guideline 406, the LLNA: DA does not require that challenge-induced dermal hypersensitivity reactions be elicited. Despite the advantages of the LLNA: DA over B.6 and OECD Test Guideline 406 (14), there are certain limitations that may necessitate the use of B.6 or OECD Test Guideline 406 (e.g. the testing of certain metals, false positive findings with certain skin irritants (such as some surfactant-type substances) (6) (1 and Chapter B.42 in this Annex), solubility of the test substance). In addition, chemical classes or substances containing functional groups shown to act as potential confounders (16) may necessitate the use of guinea pig tests (i.e. B.6; OECD Test Guideline 406 (14)). Limitations that have been identified for the LLNA (1 and Chapter B.42 in this Annex) have been recommended to apply also to the LLNA: DA (10). Additionally, the use of the LLNA: DA might not be appropriate for testing substances that affect ATP levels (e.g. substances that function as ATP inhibitors) or those that affect the accurate measurement of intracellular ATP (e.g. presence of ATP degrading enzymes, presence of extracellular ATP in the lymph node). Other than such identified limitations, the LLNA: DA should be applicable for testing any substances unless there are properties associated with these substances that may interfere with the accuracy of the LLNA: DA. In addition, consideration should be given to the possibility of borderline positive results when Stimulation Index (SI) values between 1,8 and 2,5 are obtained (see paragraphs 31-32). This is based on the validation database of 44 substances using an SI ≥ 1,8 (see paragraph 6) for which the LLNA: DA correctly identified all 32 LLNA sensitisers, but incorrectly identified three of 12 LLNA non-sensitisers with SI values between 1,8 and 2,5 (i.e. borderline positive) (10). However, as the same dataset was used for setting the SI-values and calculating the predictive properties of the test, the stated results may be an over-estimation of the real predictive properties.

PRINCIPLE OF THE TEST METHOD

• 6.The basic principle underlying the LLNA: DA 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 SI, is determined, and should be ≥ 1,8 before further evaluation of the test substance as a potential skin sensitiser is warranted. The procedures described here are based on the use of measuring ATP content by bioluminescence (known to correlate with living cell number) (17) to indicate an increased number of proliferating cells in the draining auricular lymph nodes (18) (19). The bioluminescent method utilises the luciferase enzyme to catalyse the formation of light from ATP and luciferin according to the following reaction:

• ATP + Luciferin + O 2 Image omitted; see official source Oxyluciferin + AMP + PPi + CO 2 + Light

• The emitted light intensity is linearly related to the ATP concentration and is measured using a luminometer. The luciferin-luciferase assay is a sensitive method for ATP quantitation used in a wide variety of applications (20).

DESCRIPTION OF THE ASSAY

Selection of animal species

• 7.The mouse is the species of choice for this test. Validation studies for the LLNA: DA were conducted exclusively with the CBA/J strain, which is therefore considered the preferred strain (12) (13). Young adult female mice, 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: DA response do not exist.

Housing and feeding conditions

• 8.Mice should be group-housed (21), 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 to 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. Some regulatory authorities also require a PC for each study and therefore users are encouraged to consult the relevant authorities prior to conducting the LLNA: DA. 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: DA response at an exposure level expected to give an increase in the SI ≥ 1,8 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 (e.g. SI > 10 would be considered excessive). Preferred PC are 25 % hexyl cinnamic aldehyde (Chemical Abstracts Service (CAS) number 101-86-0) and 25 % eugenol (CAS number 97-53-0) in acetone: olive oil (4:1, v/v). 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: DA regularly (i.e. conduct the LLNA: DA 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: DA 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: DA (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 (22).

• 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 (23). 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 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 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: DA;

• — 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 (2) and (24). 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 (24) (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 (6) 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 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 33). In addition, evaluating the possibility of reducing the number of mice in the PC group is only feasible when individual animal data are collected (22). Further, some regulatory authorities require the collection of individual animal data. Regular collection of individual animal data provides an animal welfare advantage by avoiding duplicate testing that would be necessary if the test substance results originally collected in one manner (e.g. via pooled animal data) were to be considered later by regulatory authorities with other requirements (e.g. individual 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: DA. The purpose of the pre-screen test is to provide guidance for selecting the maximum dose level to use in the main LLNA: DA 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.