Abstract for TestSmart--A Humane and Efficient Approach to Screening Information Data Sets (SIDS) Data

TestSmart Approach to HPV Endpoints

Sidney Green¹, Alan Goldberg², and Joanne Zurlo²
¹Howard University, Washington, D.C.; ²Johns Hopkins Center for Alternatives to Animal Testing, Baltimore, MD

At the January 26th, 1999 Vision 20/20 TestSmart Workshop, we presented a list of what we considered as alternative tests to the SIDS battery. Alternatives were defined as either refinement, reduction or replacement. Suggestions as to the status of those alternatives, i.e., whether they were ready for immediate use, needed validational studies or whether they were in the research/developmental stage, were also offered. Three breakout groups were formed, one for each of the following areas:

acute and repeat dose studies;
genetic toxicology and
reproductive/developmental.

These groups were asked to evaluate the status of alternatives in these areas and make additional suggestions and also provide an idea of the status of the alternatives that would be proposed. The information presented is an update of what was presented on January 26th, reflecting the recommendations of the breakout groups and workshop participants.

For acute toxicity, (LD₅₀), the only additional test suggested as an alternative, was the acute toxic class. Most of the methods were thought to be immediately available (see Figure 1), with the exception of the basal cytotoxicity and neutral red uptake assays. Both were thought to need some metabolic activation capabilities which then would require some degree of validation. For acute toxicity, (non-LD₅₀), in which emphasis is placed on morbidity and moribundity rather than the LD₅₀ per se, (see Figure 2), all methods were thought to be in the research/developmental stage and there were no changes from the January 26^th presentation. Target organ toxicity would be the focus in this instance, thus cultures of cells of major organs would be one manner of acquiring such information. It was recognized that more knowledge and research need to be accomplished before this approach can move to validation and ultimately to immediate use.

Very similar suggestions as those for acute toxicity, non LD₅₀ tests, were made for repeat-dose toxicity testing (see Figure 3). Research followed by validation need to be accomplished in this area as well. Difficulties are recognized, in that dose duration and frequency may be exceptionally problematic to model in an in vitro system.

Genetic toxicology represented an area in which alternatives already exist. The Ames test, mouse lymphoma and Chinese hamster ovary (CHO) cells are used routinely for gene mutations (Ames and lymphoma), and for chromosomal mutations (CHO), (see Figure 4). The in vitro micronucleus is currently undergoing validation in Japan, Europe and in the US. The Syrian hamster embryo assay has been added based on suggestions from the workshop participants. It was thought information regarding potential carcinogenicity could be useful at the screening stage.

For developmental toxicity (see Figure 5), Zebra fish has been added since the meeting in January and so has Gene chip technology (see Figure 6). The Zebra fish method is undergoing validation in Europe. Most of the methods in the developmental toxicity area are undergoing validation with the exception of gene chip techonology which is at the research stage.

It seems that reproduction looms as the most difficult area in the development of alternatives (see Figure 7). Only sperm motility and morphology are currently in use, with all other assays in the research stage. Receptor binding assays have been added since January, primarily due to the efforts with respect to endocrine disruptors.

Finally, transgenic animals have been mentioned as alternatives. It is not entirely clear as to where these methods fit, i.e., genetic toxicology, or as a means to assess mechanisms of acute non-LD₅₀ or repeat-dose toxicity testing. These studies are in the developmental stage, but are expected in the near future to be useful as screening endpoints.

Figure 1: Toxicology Data for Acute toxicity LD₅₀ (OECD 401)

Alternative Test(s)	Status
	Imm.	Valid.	Res.
basal cytotoxicity		x
up and down proced.	x
fixed dose proced.	x
limit test	x
neutral red uptake	x
acute toxic class	x

Figure 2: Toxicology Data for Acute Toxicity; non LD₅₀

Alternative Test(s)	Status
	Imm.	Valid.	Res.
cell cultures of major organ systems, e.g. vascular			x
cardiac, respiratory			x
CNS (autonomic)			x
kidney, mitochondrial			x

Figure 3: Toxicology Data for Repeat Dose Toxicity; 90 day w/eval. of testes (OECD 408) or 28 day study(OECD 407)

Alternative Test(s)	Status
	Imm.	Valid.	Res.
in vitro liver, reproductive			x
kidney, hematopoietic, CNS			x
cardiovascular, respiratory			x
Other endpoints; rate of protein synthesis, cell signaling, apoptosis			x

Figure 4: Toxicology Data for Genetic Toxicity

Alternative Test(s)	Status
	Imm.	Valid.	Res.
Ames; bact. gene mutat.	x
Mouse lymphoma; non bact. gene mutat.	x
CHO; non bact.cytogenetic assay	x
In vitro micronucleus		x
SHE transformation		x

Figure 5: Toxicology Data for Developmental Toxicity (OECD 414)

Alternative Test(s)	Status
	Imm.	Valid.	Res.
FETAX		x
whole embryo culture		x
rodent limb bud culture		x
embryonic stem cells		x
zebra fish		x

Figure 6: Toxicology Data for Developmental Toxicity (OECD 414)

Alternative Test(s)	Status
	Imm.	Valid.	Res.
Gene chip technology (analysis of receptors, gene activators, enzymes ets.)			x

Figure 7: Toxicology Data for Toxicity to Reproduction (OECD 421 or 422)

Alternative Test(s)	Status
	Imm.	Valid.	Res.
sertoli cell lines			x
sperm motility	x
sperm morphology	x
sperm penetration			x
cultured primary follicles			x
receptor binding assays			x

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