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ANALYTICAL AND OTHER METHODS OF TOXICOLOGY TOXICITY TESTING

Toxicity testing is conducted for the purpose of human health risk assessment

Risk assessment:

Quantitative estimation of the probability of delirious effects under given exposure

condition

Toxicity testing are not designed to demonstrate the safety of a chemical, but to characterize

the toxic effects that chemical produces.

Any chemical that is being developed for possible Introduction into commerce is subjected to

toxicity testing to satisfy the regulation of one or more regulatory agencies.

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NOAEL: no observed adverse effect level LOAEL: lowest observed (observable) adverse effect level

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ACUTE DERMAL TOXICITY TESTS

DESCRIPTION

All products for human and veterinary consumption are regulated for evaluation for

toxicology testing. With dermal toxicity studies, rabbits serve as standards for evaluating

local toxicity (irritation). Guinea pigs are generally listed in testing guidelines as an

acceptable species, but their use is uncommon for dermal toxicology evaluations. They have,

until recently, been the species of choice for sensitization studies.

PRIMARY IRRITATION

Dermal acute studies are designed to provide information on local effects, particularly skin

irritation and corrosion. In the aftermath of World War II and the development of chemical

warfare research, the need to ensure the safety of consumer products generated significant

interest.

The rabbit is the species of choice for dermal irritation because it has relatively sensitive skin

as compared to human skin. This species is also easier to handle than larger species and

rabbit skin has high permeability. Because of the rabbits enhanced sensitivity to dermal

insult, however, it is generally considered to be over-predictive of human irritation, thus

bringing into question its relevance to human risk assessment. Many current regulations

specify albino rats as preferable to rabbits although rabbits still represent the standard for

local toxicity (irritation) evaluations. The guinea pig is generally listed in testing guidelines

as an acceptable species, but is rarely used in dermal irritation toxicity evaluations.

Draize et al. (1944) published a quantitative assessment of skin irritation as a guideline for

product safety. They defined a primary local irritant as a substance that produced an

inflammatory dermal reaction. The inflammatory process as it pertained to dermal irritation

was characterized by the presence of edema* and erythema. Table 8.1 outlines the grading

of primary reactions of rabbit skin based on Draize observations. Because of the potential for

pain and skin damage resulting from cutaneously applied materials, careful monitoring of

cutaneous irritation and toxicity studies is now mandated by animal welfare regulations.

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Study Design and Procedures

Dermal irritation studies are designed to mimic human exposure and are generally performed

on one to three albino rabbits. A test area is prepared by shaving the fur(2 to 3 cm ) on the

back or abdomen. Initially, a single animal is exposed sequentially (3 min and 1 and 4 hr) to

the test material (0.5 ml or 0.5 g). Most drugs and biological materials are left uncovered.

Applications may be covered if a material is applied under a bandage (burn treatment) or

covered with clothing (to mimic occupational exposure to chemicals).

A semi-occlusive covering generally consists of a porous dressing such as gauze held in

place with non-irritating tape. If dermal corrosion is seen after exposure, the test is terminated

and the material is classified as corrosive.

If no corrosion is seen, two additional animals are exposed to the material for up to 4 hr and

irritation is scored according to the Draize irritation potential classification, as detailed in

Table 8.2. The sum of the mean erythema and edema scores is computed and listed as the

primary dermal irritation index (PDII).

SKIN SENSITIZATION

Evaluation of the potential to produce sensitization is required for many chemicals.

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Upon application to and penetration of the skin, a chemical can elicit a subsequent reaction,

usually days or weeks after repeated exposures. This type of skin sensitizationis known as

allergic contact sensitivity or dermatitis. The response requires at least 24 to 72 hr for

development and a reaction is characterized as a type IV (delayed-type hypersensitivity) cell-

mediated immunity. Responses range from mild irritation resulting in erythema and

induration, to the development of eczema, eruptions, and flaring, to full systemic immune

manifestations. The response to a chemical is typically dosedependent and self-limiting (days

to weeks) and of varying intensity. Furthermore, the process requires initial sensitization,

adaptation, and subsequent re-challenge.

Study Design and Procedures

Invariably, the test animals used for skin sensitization studies are guinea pigs, particularly

because of their known susceptibility to a variety of chemical sensitizers.

Consequently, the most common skin sensitization methods include the guinea pig

maximization test (GPMT) of Magnusson and Kligman and the Buehler test. Studies utilize

10 to 20 animals in a treated group and 5 to 10 in a control group. For the GPMT, induction is

started according to the adjuvant method by a set of intradermal injections (test article,

Freunds adjuvant, and test article in adjuvant), followed 1 wk later by topical application,

and topical challenge 2 wk later.

PHOTOALLERGIC AND PHOTOTOXIC REACTIONS

Dermal phototoxicity of a chemical is defined as a toxic response elicited after dermal or

systemic exposure to a chemical following subsequent exposure to light, particularly

ultraviolet (UV) radiation from sunlight. The biological effects of UV rays alone are of lower

frequency and longer wavelength (about 108 to 106m) than ionizing radiation (107 to 10

12 m for x-rays). Thus the effects of UV radiation are less penetrating and benign.

Unlike ionizing radiation, skin damage induced by UV rays is mediated principally by the

generation of reactive oxygen species (ROS) and the interruption of melanin production. Like

ionizing radiation, however, cumulative or intense exposure to UV rays precipitates DNA

mutations: base pair insertions, deletions, single strand breaks, and DNA-protein cross-links.

DNA repair mechanisms play an important role in correcting UV-induced DNA damage and

in preventing further consequences of excessive UV exposure (as with sunburn).

ACUTE OCULAR TOXICITY TESTS

The Draize eye test, however, still remains the only official, regulatory-approved procedure

for predicting ocular irritants. With the development of alternative non-animal procedures to

replace the Draize test, the data generated in the Draize procedure are also used as standards

against which the performances of alternative methods are measured.

EYE IRRITATION AND CORROSION TESTING

The Draize test involves a standardized protocol for instilling agents onto the corneas and

conjunctivae of laboratory animals, particularly rabbits. A sum of ordinal-scale items of the

outer eye gives an index of ocular morbidity. The test uses a scoring system that describes

reactions produced in the cornea, conjunctiva, and iris. Since its inception, the test has

undergone several modifications, including the addition of other descriptive parameters such

as thickness of the eyelids, induction of erythema, edema, discharge, corneal opacity,

capillary damage, and pannus (vascularization) of the cornea.