Measurement of the Toxicity of Adjuvants

3.3.1. Cytotoxicity Assay in Cultured Monolayers of Human or Animal Cell Lines

This type of assay has the great merit of reducing the number of animals which must be used to comply with standardized toxicity tests. The MTT assay (13) was adapted for determining cell survival and proliferation by a number of workers (14-16) with different cell types and toxins.The assay compares favorably with other similar systems (17), is less time-consuming and objective than microscopic examination of cells, and eliminates the risks associated with assays involving radioisotope release. As examples, the protocols for three different cell lines have been described, but these are not exclusive.

1. Tissue culture cells and growth conditions. The cells should be checked for the absence of virus contamination. This is confirmed by electron microscopy and for the presence of contaminating mycoplasmas by a specific staining technique before use. Human colon adenocarcinoma (CaCO-2) cells are grown in Eagle's MEM medium with Earle's salts and 25 mM HEPES (Gibco) in 80-cm2 tissue-culture flasks (Falcon). Non-essential amino acids (1.0% w/v), glutamine (2 mmol/L), 100 |g/mL penicillin/streptomycin, 1.0% v/v of growth promoter, insulin-transferrin-selenium, and 10% v/v fetal calf serum are added and the cells are incubated at 37°C in 5.0% carbon dioxide atmosphere. The cells are routinely split 1 in 5 by rinsing with 5.0 mL sterile PBS followed by 2.0 mL of 0.25% w/v trypsin/EDTA. A confluent monolayer of CaCO-2 cells in an 80.0-cm2 flask is obtained usually within 5-7 d, with regular changes of medium. A cell suspension of cells prepared by trypsinization is used to inoculate wells in a 24-well plate.

African green monkey kidney (VERO) or HeLa cells, are grown in Eagle's MEM medium (Gibco) containing 100 |g/mL penicillin/streptomycin and 5.0% v/v fetal calf serum. The cells are incubated at 37°C in an atmosphere of 5% CO2. VERO and HeLa cells are routinely split in the same manner as the CaCO-2 cells.

2. HeLa or VERO cells are harvested with trypsin/EDTA and resuspended in growth medium to a density of 5 x 104 cells/mL. Each well of a flat-bottomed 24-well plate is loaded with 200 |L of the cell suspension and incubated at 37°C overnight. The growth medium (100 |L) is discarded and 100 |L of twofold dilutions of the adjuvant in tissue-culture medium is added to each well: duplicate wells of each dilution are set up. Cells with PBS only or 1.0% v/v Triton X-100 in PBS serve as the 100% and 0% live controls, respectively. After 24 h incubation at 37°C, 20 |L of MTT solution (5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (Sigma) in PBS, filter-sterilized are added to each well and incubation continued for 4 h. After emptying the wells the resultant formazan crystals are solubilized by the addition to each well of 100 ||L 0.4 mol/L HCl in dimethyl sulphoxide v/v (DMSO; Sigma) and the absorbance measured at 540 nm in the Anthos 2001 plate reader. The percentage of cell deaths, adopted to take account of the variable growth of the cells, was calculated with the formula:

[A540nm test - A540nmTriton X +ve Control]

[A540nm PBS -ve control - A540nmTriton X +ve Control]

3.3.2. Intracutaneous Toxicity Test

This procedure assesses any skin irritation at the site of injection and is based on the British Standard 5736: Part 7 (18). This test may be relevant with some adjuvants, which are to be included in vaccines injected by the intradermal or subcutaneous routes. For example, Kensil et al. (19) fractionated Quil A from Quillaja saponaria, the South American soap tree, and one preparation QS-7 was nontoxic at an intradermal dose of 500 ^g whereas QS-18 was lethal at a dose of 25.0 ^g. The latter preparation would not be acceptable either as a saponin adjuvant nor as part of any other adjuvant formulation. This type of test may involve single-dose toxicity or repeated dose toxicity reactions of the adjuvant formulation. The test is usually done by intraperitoneal (ip) or subcutaneous (sc) injection into two mammalian species, but the number of animals in the test groups is being questioned and some authorities may well invoke the 3 Rs, namely replacement, reduction, and refinement for the sake of animal welfare.

3.3.2.1. Single-Dose Toxicity Test

This is a qualitative and quantitative study of the possible toxic reactions, which may result from a single administration of the active substance, in this instance the adjuvant, in an acute toxicity test. As with other tests, it is important to use the adjuvant alone or in the injectable form. The test should be done in two mammalian species, with equal numbers of males and females, if a vet erinary product the intended animal should be included. There should be at least two routes of administration, for example by ip and sc injection. After injection, the animals should be examined at regular intervals, at least three times daily, for not less than 7 d, and any animal with obvious signs of ill health or in a moribund state should be killed.

3.3.2.2. Repeated-Dose Toxicity Test

This is intended to monitor the effect of repeated administration of vaccines containing an adjuvant component. It is the responsibility of the investigator to give valid reasons for the extent and duration of the trials and the dosages chosen. However, the maximum dose should be selected so as to indicate potential harmful effects and lower doses will enable the animal's tolerance to the new adjuvant. The repeated-dose toxicity test should be done in two mammalian species (1 nonrodent).

Animals that are mentioned in European rules governing medicinal products (28) for use in these two tests are: mouse (Mus musculus), rat (Rattus norvegicus), guinea-pig (Cavia porcellus), golden hamster (Mesocricetus auratus), rabbit (Oryctolagus cuniculus), nonhuman primates, dog (Canis familiaris), cat (Felis catus), quail (Coturnix coturnix).

Evaluation of the adjuvant may be done by a variety of means: monitoring the behavior and weight gain of the animals, hematological, and physiological tests. If an animal dies, an autopsy and histological examination of tissues, including the sites of injection, should be done.

1. The adjuvant is dissolved/suspended in either a polar solvent, sterile physiological saline, or a nonpolar solvent, sesame oil (Ph. Eur), usually heated at 180°C for 60 min.

2. Preparation of animals. The fur is clipped on the back of each animal, e.g., rabbits, before injection.

3. Rabbit 1. (a) inject four sites on the left-hand side of the body with 0.1 mL of the test mixture subcutaneously or (b) inject four sites on the right-hand side with 0.01 mL intradermally with: (i) adjuvant in polar solvent; (ii) polar solvent alone; (iii) adjuvant in nonpolar solvent; iv) nonpolar solvent alone. The injection sites are examined for 5 d and the size of any skin reactions measured with precision calipers, for example, Mecanic in nylon-asbestos (Camlab, U.K.).

4. Rabbits 2, 3, and 4 should only be injected if the rabbit 1 test is negative.

5. The injection sites are examined for erythema (redness at the site of injection), eschar (scab formation at the site of injection), or edema (swelling at the injection site) (Table 1).

3.3.3. Systemic Toxicity Test

The aim of this procedure is to measure undesirable effect(s) at sites distant from the injection site, which may become apparent after the administration of

Table 1

Classification System for Skin Reactions

Reaction Numerical grading

Erythema and eschar formation:

No erythema 0

Very slight erythema 1

Well-defined erythema 2

Moderate to severe erythema 3

Severe erythema (beet-redness) to slight 4 eschar formation Edema formation:

No edema 0

Very slight edema 1

Well-defined edema (edges of area well 2

defined by definite raising)

Moderate edema (raised approximately 1 mm) 3

Severe edema (raised more than 1 mm 4 and extending beyond exposure area)

NOTE: Other adverse changes at the skin sites should be recorded and reported.

the adjuvant alone or the adjuvant formulation. The adjuvant is injected intra-peritoneally in polar or nonpolar diluents or intravenously in a nonpolar solvent with appropriate controls. These are tests which the regulatory bodies may require with groups of five mice, but there may be moves to reduce the number of animals to be tested. It is feasible that these tests could eventually be phased out when there are sufficient experimental results accumulated to allow the validation of alternative toxicity tests. The method is based on British Standard 5736: Part 3 (20).

1. Groups of five weanling mice, 3-4-wk old are weighed and injected, either intra-peritoneally with 0.5-mL volumes of graded doses of the adjuvant mixtures:

Group 1. Adjuvant in sesame oil Group 2. Sesame oil alone Group 3. Adjuvant in physiological saline Group 4. Physiological saline alone Or, intravenously with 1.0 mL of:

Group 5. Adjuvant in physiological saline Group 6. Physiological saline alone

2. The animals are observed for 14 d, frequently during the 4 h immediately following injection and at least three times a day thereafter.

3. Record any visible signs of reaction after injection of the adjuvant preparation, for example, time of onset after injection, their duration, and intensity. Weigh all the animals daily for 7 d, refer to the "weight-gain test" below, and kill all surviving animals and record the appearance of the animals. Record any deaths if they occur on the respective day after injection. Postmortem all animals at the end of the experiment and record the appearance of organs and the histological examination of tissues of interest including: heart, lungs, gastrointestinal tract, liver, spleen, kidneys, and gonads. The report could be produced in the format shown in Table 2.

3.3.4. Mouse Weight-Gain Test

The 7-d mouse weight-gain test is still a standard and reliable method. After injection of a substance containing endotoxin, the animal may show a decrease in weight during 24 h if endotoxin is present (21). If this is followed by a steady increase in the animal's weight over 7 d, it is assumed that the product is acceptable. If, on the other hand, the product is highly toxic the animal may steadily continue to lose weight or in extreme cases become moribund and is killed. The tests in Subheading 3.3.2. and 3.3.3. may provide evidence of unacceptable levels of toxicity in which case it may be unnecessary to proceed with a weight-gain test as the adjuvant is probably too reactive.

The protocols for these laboratory assays should not be regarded as alternatives to statutory tests required for licensed medical or veterinary products, however, they will show whether financial investment in a new immuno-potentiator or adjuvant formulation is warranted. Invariably, if a new adjuvant formulation gives a positive reaction in one of the tests described above, it is highly unlikely that the preparation will be suitable for routine vaccine use (see Note 2).

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