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Protocol no. 22
TETRAHYMENA THERMOPHILA OCULAR IRRITANCY TEST
The cytotoxic effect of chemicals upon the protozoan,
Tetrahymena thermophila, in culture is assessed by studying their effect
upon the normal motility of the organism. It is suggested that the Tetrahymena
thermophila test may be useful as part of a small battery of in vitro tests
in the evaluation of the ocular irritancy potential of chemicals.
CONTACT
Dr Jerald Silverman, D.V.M. Assistant Vice-President
for Research Animal Facilities Director, Master of Laboratory Animal Science
Program Hahnemann University Broad & Vine Mail Stop 436 Philadelphia,
PA 19102-1192 USA Telephone: USA - 215-762-7970 Fax: USA - 215-762-7449
RATIONALE
The basis of this test is that a cytotoxic chemical
(regardless of site or mechanism of action) will interfere with the normal
motility of the protozoan, Tetrahymena thermophila, in culture. The degree
of interference of motility as compared to control cultures, related to
the concentration of the test compound, provides an indication of toxicity.
Tetrahymena thermophila would appear to be a suitable organism for use
in the development of an alternative to the Draize ocular irritancy test,
since it is: a) a typical eukaryotic cell b) well characterised c) a commonly
found freshwater ciliated protozoan d) has for many years been used in
biological indicator systems.
BASIC PROCEDURE
The protozoan, Tetrahymena thermophila, is maintained
in culture and exposed to test compounds over a range of concentrations.
The cells are exposed to test compound for 2 minutes before being examined
microscopically, and an assessment made of their motility, in comparison
to control cultures. The reciprocal of the approximate chemical dilution
which allows at least 90% of the cells to move in a relatively normal manner
(i.e. highest tolerated dose, HTD) is determined. The lower the resulting
HTD value, the lower the irritancy potential of the compound.
CRITICAL ASSESSMENT
This is a relatively low cost, simple and rapid
test based on standard protozoan culture techniques. Indeed, this procedure
is one of the more rapid in vitro test procedures. This is because of the
relatively short time periods required to both culture the organism from
stock, and expose the cells to test chemical e.g only 2 days are needed
to prepare the test organism from stock cultures; 2 minutes for exposure
to test chemical; and only a few seconds for the endpoint, cell counting.
Optimum test conditions The authors have studied the optimum test conditions
required for this test (Silverman, 1987). Since the variables studied ("inbred"
vs. "outbred" strains, incubation temperature, culture age, effect
of growth surface area, etc.) did not significantly change the results
found in this test system, the authors recommend the routine use of: 1.
the "inbred" strain 30377 of Tetrahymena thermophila 2. culture
in the dark at RT 3. growth of cells in standard screw-top test tubes 4.
growth of cells for 48 hours (logarithmic growth phase) prior to testing.
TEST STATUS
Undergoing in-house development, as well as evaluation
as a potential in vitro alternative to the Draize rabbit eye irritancy
test, in phases I, II and III of the validation scheme conducted by the
Soap and Detergent Association (SDA) of the United States. This test has
also been evaluated in phases I and II of the validation scheme currently
being conducted by the Cosmetic, Toiletries and Fragrance Association (CTFA)
of the United States, as well as the MEIC (Multicentre Evaluation of In
Vitro Cytotoxicity) scheme being conducted by the Scandinavian Society
for Cell Toxicology. It is also being used by several commercial organisations.
Eight coded chemicals were studied in phase I of the SDA scheme. Results
showed that the Tetrahymena test was able to rank all the test samples
in order when compared to in vivo testing results. As measured by standard
correlation statistics, the following results were generated: rank correlation
was +0.69 with Spearman's Rho (maximum of +0.75) and +0.57 with Kendall's
tau (maximum of 0.57). Pearson's product moment correlation coefficient
was +0.88 (maximum of +0.88). ORGANISATIONS USING THE TEST The test is
currently being used in several commercial laboratories, including pharmaceutical,
cosmetic and commercial testing laboratories, throughout the United States.
CHEMICALS TESTED Acetic acid (3%) Acetone Benzalkonium chloride (0.1%,
1%) n-Butanol Ethanol (95%) Ethylene glycol Ethylene glycol diethyl ether
Isopropanol (15%, 70%) Isopropyl myristate Methylethyl ketone Methylisobutyl
ketone Miranol 2 MCA MOD (38%) Propylene glycol Quaternium 23 (50%) Silver
nitrate (1%) Sodium hydroxide (0.5%, 1%) Sodium lauryl sulphate (10%) Triethanolamine
lauryl sulphate (neat, 5%) Tween 20 Hydrochloric acid (0.05N, 0.1N, 0.2N)
REFERENCES
- Kay, J. & Calandra, J. (1962) Interpretation
of eye irritation tests. J. Soc. Cosmet. Chem., 13, 281.
- Silverman, J. (1983) Preliminary findings on
the use of protozoa (Tetrahymena thermophila) as models for ocular irritation
testing in rabbits. Lab. Animal Sci., 33, 56-59.
- Silverman, J. & Pennisi, S. (1987) Evaluation
of Tetrahymena thermophila as an in vitro alternative to ocular irritation
studies in rabbits. J. Toxicol.-Cut. & Ocular Toxicol., 6(1), 33-42.
IP-22 © June 1991
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