(paragraphs 25(c) and 29(c))
PERMISSIBLE LIMITS OF TOXICITY
1. (1) For the purposes of paragraphs 25(c) and 29(c) of these Regulations, a substance is excessively toxic for humans in all of the following cases:
(a) its acute oral LD50 value for rat is 5 g or less per kg of body weight;
(b) its acute dermal LD50 value for rabbit is 2 g or less per kg of body weight; and
(c) its LC50 value for a one-hour exposure, determined using rats, is 20,000 ppm by volume of gas or vapour or less, or 200 mg/L by volume of mist or dust or less, if gas, vapour, mist or dust is likely to be encountered when the substance is used in a reasonably foreseeable manner.
(2) LD50 values are to be determined in conformity with good toxicological practice.
(3) The number of deaths during a 14-day period after dosage is to be used as the basis for calculation of the LD50 value.
(4) A sufficient number of animals are to be used to give a statistically significant result. The result is to be calculated using methods based on good statistical practice.
(5) The methods referred to in the following publications, and other methods giving similar results, are acceptable:
(a) C.I. Bliss, “The determination of the dosage-mortality curve from small numbers”, Quarterly Journal of Pharmacy and Pharmacology, 1938, Volume 11, page 192; and
(b) J.T. Litchfield and W.F. Wilcoxon, “A simplified method of evaluating dose-effect experiments”, Journal of Pharmacology and Experimental Therapeutics, 1949, Volume 96, page 99.
METHOD FOR DETERMINING DERMAL LD50 VALUE
2. The dermal LD50 value is to be determined in the following manner:
(a) Prepare the animals by clipping the skin of the trunk free of hair. For approximately one half of the animals, make epidermal abrasions every 2 cm (0.8 inches) or 3 cm (1.2 inches) longitudinally over the area of exposure. Ensure that the abrasions are sufficiently deep to penetrate the stratum corneum (horny layer of the epidermis), but not so deep as to disturb the derma — that is, not to produce bleeding.
(b) In the acute exposures, hold the agent in contact with the skin by means of a sleeve for periods varying up to 24 hours. Use a sleeve made of rubber dam or other impervious material so that the sleeve has ends reinforced with additional strips and fits snugly around the trunk of the animal. Tuck the ends of the sleeve to permit the central portion to “balloon” and furnish a reservoir for the dose. Ensure that the reservoir has sufficient capacity to contain the dose without pressure. The dimensions of the sleeves and the approximate body surface exposed to the test substance are set out in the following table.
Diameter at ends Overall length Range of weight of animals Average area of exposure Average percentage of total body surface 7.0 cm (2.8 inches) 12.5 cm (4.9 inches) 2 500 g-3 500 g (5.5 pounds-7.7 pounds) 240 cm2 (37.2 inches2) 10.7
(c) Vary the size of the sleeves to accommodate smaller or larger subjects. Mesh wire screen may be used instead of the sleeve in the testing of unctuous materials that adhere readily to the skin. Pad and raise the screen approximately 2 cm (0.8 inches) from the exposed skin. In the case of dry powder preparations, moisten the skin and substance with physiological saline before exposure. Slip the sleeve or screen over the gauze which holds the dose applied to the skin. In the case of finely divided powders, distribute the measured dose evenly on cotton gauze and attach the gauze to the area of exposure.
(d) Slip the sleeve onto the animal. Place the animal in a comfortable but immobilized position in a multiple animal holder. Introduce selected doses of liquids and solutions under the sleeve. Collect and reapply any slight leakage from the sleeve which may occur during the first few hours of exposure. Adjust dosage levels in subsequent exposures, if necessary, to enable a calculation of a dose that would be fatal to 50% of the animals. This can be determined from mortality ratios that are obtained based on the various doses that are used. At the end of 24 hours, remove the sleeves or screens, measure the volume of any unabsorbed material and note the skin reactions. Clean the subjects by thorough wiping, observe them for gross symptoms of poisoning and then observe them for two weeks.
3. For the purposes of this Schedule, “LD50” means the dose that will kill 50% of test animals under the specified conditions of the test and “LC50” means the concentration of gas and vapour that will kill 50% of test animals under the specified conditions of the test.
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