**13.0 GLOSSARY**[^1]

**Accuracy**[^2]**:** (a) The closeness of agreement between a test
method result and an accepted reference value. (b) The proportion of
correct outcomes of a test method. It is a measure of test method
performance and one aspect of "relevance". Accuracy is highly dependent
on the prevalence of positives in the population being examined.

**Acute Toxic Class (ATC) method:** An acute oral systemic toxicity test
method based on testing groups of animals at fixed doses in a sequential
manner. The lethality outcomes are used to classify a test substance
into the appropriate GHS acute oral toxicity category.

**Adjusted R^2^:** R^2^ values that are adjusted for the relative
proportion of data points to explanatory variables. Adjusted R^2^ = 1 -
(1 - R^2^)\[(n -- 1)/(n - k - 1)\] where k = number of independent
variables and n = number of observations. See "coefficient of
determination."

**ANOVA:** One-way (and two-way) analysis of variance. ANOVA compares
the measurements (continuous variables) of three or more groups when the
data are categorized in one way (one-way) or two ways (two-way). ANOVA
assumes that the populations compared are normally distributed and that
the variances for the groups to be compared are approximately equal.

**Assay****^2^:** The experimental system used. Often used
interchangeably with "test" and "test method."

**Biphasic dose-response:** Dose-response in which cytotoxicity
increases (as dose increases), plateaus, and then increases again. See
**Section 2.6.3**.

**Category prediction:** The GHS hazard category that includes the
predicted LD~50~ value for a test chemical.

**Coded substances:** Substances labeled by code rather than name so
that they can be tested and evaluated without knowledge of their
identity or anticipation of test results. Coded substances are used to
avoid intentional or unintentional bias when evaluating laboratory or
test method performance.

**Coefficient of variation:** A statistical representation of the
precision of a test. It is expressed as a percentage and is calculated
as follows:

> $$\begin{array}{r}
> \begin{array}{r}
> \left( \underline{\text{standard}\text{deviation}} \middle| \right) \\
>
> \end{array} \\
>
> \end{array}$$

**Coefficient of determination:** In linear regression, it denotes the
proportion of the variance in Y and X that is shared. Its value ranges
between zero and one and it is commonly called called "R^2^." For
example, R^2^ = 0.45, indicates that 45% of the variance in Y can be
explained by the variation in X and that 45% of the variance in X can be
explained by the variation in Y.

**Concordance^2^:** The proportion of all substances tested that are
correctly classified as positive or negative. It is a measure of test
method performance and one aspect of "relevance." The term is often used
interchangeably with "accuracy." Concordance is highly dependent on the
prevalence of positives in the population being examined. In the
NICEATM/ECVAM study, concordance was used to describe the proportion of
test substances that were correctly classified into GHS acute oral
toxicity hazard categories, or to describe the proportion of test
substances for which the laboratories obtained the same classification
result.

**Confluency:** A state in which cells in culture come into contact with
other cells in the same culture to form a complete sheet of cells
(monolayer). For this study, confluency is determined as a percentage of
cell coverage of the tissue culture vessel growth surface (e.g., cell
monolayer has 80% confluency).

**Cytotoxicity:** The adverse effects resulting from interference with
structures and/or processes essential for cell survival, proliferation,
and/or function. For most chemicals, toxicity is a consequence of
non-specific alterations in \"basal cell functions\" (i.e., via
mitochondria, plasma membrane integrity, etc.), which may then lead to
effects on organ-specific functions and/or death of the organism. These
effects may involve the integrity of membranes and the cytoskeleton,
cellular metabolism, the synthesis and degradation or release of
cellular constituents or products, ion regulation, and cell division.

**Definitive test:** The main test of the cytotoxicity assay for
determining the IC~50~. The concentration closest to the range finder
test IC~50~ serves as the midpoint of the concentrations tested in a
definitive test. Compared to the range finder test, the definitive test
uses a smaller dilution factor for the concentrations tested.

**Discordant chemicals:** Chemicals for which the LD~50~ is not
accurately predicted by the IC~50~ (and the associated regression
formula) or the GHS toxicity category is not accurately predicted by the
IC~50~ (and the associated regression formula). Also referred to as
"outliers."

**EDIT:** Evaluation-guided Development of New *In vitro* Test
Batteries. An international project coordinated by the Scandinavian
Society for Cell Culture to develop new *in vitro* tests for toxicity
and toxicokinetics to be incorporated into test batteries for predicting
acute and chronic systemic toxicity.

**Endpoint^2^:** The biological process, response, or effect assessed by
a test method.

**Fixed Dose Procedure (FDP):** An acute oral systemic toxicity test
method based on testing groups of animals at fixed doses. Evident
toxicity outcomes are used to classify a test substance into the
appropriate GHS acute oral toxicity category.

**F~G~:** An empirical factor for the RC regression line that represents
the expected precision of LD~50~ predictions from basal cytotoxicity
data. The LD~50~ values of 73% of the 347 RC chemicals are localized in
the dose range around the RC regression line by **F~G~** ≤ log 5. The
factor represents the expected difference between the LD~50~ determined
in animal experiments and the LD~50~ estimated from the IC~50~ on the RC
regression line.

**Geometric mean:** The antilog of the mean of the logarithm of the
values. It is less affected by extreme values than the arithmetic mean.

**Globally Harmonized System (GHS):** A classification system presented
by the United Nations that provides (a) a harmonized criteria for
classifying substances and mixtures according to their health,
environmental and physical hazards, and (b) a harmonized hazard
communication elements, including requirements for labeling and safety
data sheets.

**Good Laboratory Practices (GLP)^2^:** Regulations promulgated by the
U.S. Food and Drug Administration and the U.S. Environmental Protection
Agency, and principles and procedures adopted by the Organization for
Economic Cooperation and Development and Japanese authorities that
describe record keeping and quality assurance procedures for laboratory
records that will be the basis for data submissions to national
regulatory agencies.

**Guidance Document:** *Guidance Document on Using In Vitro Data to
Estimate In Vivo Starting Doses for Acute Toxicity* (ICCVAM 2001b).

**Hazard^2^:** The potential for an adverse health or ecological effect.
A hazard potential results only if an exposure occurs that leads to the
possibility of an adverse effect being manifested.

**Hill function:** The IC~50~ values are determined from the
concentration-response using a Hill function which is a four parameter
logistic mathematical model relating the concentration of the test
chemical to the response (typically following a sigmoidal shape).

$$\begin{array}{r}
Y = \text{Bottom} + \frac{\text{Top} - \text{Bottom}}{1 + \text{10}^{(\text{logIC50} - X)\text{HillSlope}}} \\

\end{array}$$

where Y= response, X is the logarithm of dose (or concentration), Bottom
is the minimum response, Top is the maximum response, logIC~50~ is
logarithm of X at the response midway between Top and Bottom, and
HillSlope describes the steepness of the curve.

**Hill function (revised):** Some unusual dose-responses did not fit the
Hill function well. To obtain a better model fit, the Bottom parameter
was estimated without constraints (the previous practice was to use
Bottom = 0). However, when Bottom ≠ 0, the EC~50~ reported by the Hill
function was not the same as the IC~50~ since the Hill function relies
on EC~50~ defined as the point midway between Top and Bottom. Thus, the
Hill function calculation using the Prism® software was rearranged to
calculate the concentration corresponding to the IC~50~ as follows.

$X = \text{log}\text{EC}_{\text{50}} - \frac{\text{log}\left( \frac{\text{Top} - \text{Bottom}}{Y - \text{Bottom}} - 1 \right)}{\text{HillSlope}}$

X is the logarithm of concentration at 50% response, logEC~50~ is
logarithm of concentration at the response midway between Top and
Bottom, Top is the maximum response, Bottom is the minimum response, Y =
50 (i.e., 50% response), and HillSlope describes the steepness of the
curve.

**IC~50~:** test chemical concentration producing 50% inhibition of the
endpoint measured (i.e., cell viability).

**Interlaboratory reproducibility^2^:** A measure of whether different
qualified laboratories using the same protocol and test substances can
produce qualitatively and quantitatively similar results.
Interlaboratory reproducibility is determined during the prevalidation
and validation processes and indicates the extent to which a test method
can be transferred successfully among laboratories.

**Intralaboratory repeatability^2^:** The closeness of agreement between
test results obtained within a single laboratory when the procedure is
performed on the same substance under identical conditions within a
given time period.

**Intralaboratory reproducibility^2^:** The first stage of validation; a
determination of whether qualified people within the same laboratory can
successfully replicate results using a specific test protocol at
different times.

***In vitro:*** In glass. Refers to assays that are carried out in an
artificial system (e.g., in a test tube or petri dish) and typically use
single-cell organisms, cultured cells, cell-free extracts, or purified
cellular components.

***In vivo:*** In the living organism. Refers to assays performed in
multicellular organisms.

**K~ow~**: Octanol:water partition coefficient.

**LC~50~:** Acute lethal serum or blood concentrations.

**LD~50~**: The calculated value of the oral dose that produces
lethality in 50% of test animals (rats and mice). The LD~50~ values
serve as reference values for the *in vitro* tests.

**LD~50~ (initial):** Acute oral rat and mouse LD~50~ values used during
the chemical selection process. For RC chemicals, LD~50~ values were
those used in the RC regression, which were largely from the 1983/84
RTECS®. For chemicals that were not included in the RC, the initial
LD~50~ values came from HSDB or 2002 RTECS®.

**LD~50~ (reference):** Acute oral rodent LD~50~ values from rats and
mice were located through literature searches and references from major
toxicity databases such as RTECS®. Studies were reviewed to identify the
most appropriate LD~50~ values for each chemical. Values obtained using
feral animals, preanesthetized animals, or animals less than 4 weeks of
age were not used. Values reported as inequalities were not used.
Reference LD~50~ values were determined by calculating the geometric
mean of the acceptable LD~50~ values. Data were used in generation of
the 3T3 and NHK NRU regressions.

**Maximum:minimum value**: Ratio of minimum acceptable LD~50~ to maximum
acceptable LD~50~.

**MEIC:** Multicentre Evaluation of *In Vitro* Cytotoxicity. An
international effort established by the Scandinavian Society for Cell
Toxicology and initiated in 1983 to evaluate the relationship and
relevance of *in vitro* cytotoxicity for predicting the acute toxicity
of chemicals in humans.

**Millimolar regressions:** Linear regressions with IC~50~ values in
mmol/L and LD~50~ values in mmol/kg.

**Negative control:** An untreated sample containing all components of a
test system, except the test substance solvent, which is replaced with a
known non-reactive material, such as water. This sample is processed
with test substance-treated samples and other control samples to
determine whether the solvent interacts with the test system.

**Neutral red (NR):** A weakly cationic water-soluble dye that stains
living cells by readily diffusing through the plasma membrane and
concentrating in lysosomes where it electrostatically binds to the
anionic lysosomal matrix.

**Neutral red uptake (NRU):** Concentration of neutral red dye in the
lysosomes of living cells. Altering the cell surface or the lysosomal
membrane by a toxicological agent causes lysosomal fragility and other
adverse changes that gradually become irreversible. The NRU test method
makes it possible to distinguish between viable, damaged, or dead cells
because these changes result in decreased uptake and binding of NR
measurable by optical density absorption readings in a
spectrophotometer.

**NHK:** **N**ormal **H**uman epidermal **K**eratinocytes (from neonatal
foreskin).

**Optical density (OD):** The absorption (i.e., OD measurement) of the
resulting colored solution (colorimetric endpoint) in the NRU assay
measured at 540 nm ± 10 nm in a spectrophotometric microtiter plate
reader using blanks as a reference

**Outlier:** For any measurement, an extreme value in the NICEATM/ECVAM
study was referred to as an "outlier" if it passes a statistical test
for outliers at the 99% level. With respect to chemicals, it refers to
chemicals that do not fit (using the specified criteria) an
IC~50~-LD~50~ linear regression model. It may also refer to chemicals
for which the predicted GHS toxicity category does not match the
reference *in vivo* GHS toxicity category.

**Performance^2^:** The accuracy and reliability characteristics of a
test method (see "accuracy", "reliability").

**pH:** A measure of the acidity or alkalinity of a solution. pH 7.0 is
neutral; higher pHs are alkaline, lower pHs are acidic.

**Plate reader:** A spectrophotometric device for measuring light
intensity as a function of color/wavelength (i.e., optical
density/absorption at 540 nm ± 10 nm for NRU) in 96-well microtiter
tissue culture plates.

**Positive control:** A sample containing all components of a test
system and treated with a substance known to induce a positive response,
which is processed with the test substance-treated and other control
samples to demonstrate the sensitivity of each experiment and to allow
for an assessment of variability in the conduct of the assay over time.

**Predictivity**^2^: Proportion of *in vivo* category matches for all
substances with *in vitro* predictions for a particular category.
Predictivity is an indicator of test accuracy.

**Protocol^2^:** The precise, step-by-step description of a test,
including the listing of all necessary reagents, criteria and procedures
for the evaluation of the test data.

**Quality assurance (QA)^2^:** A management process by which adherence
to laboratory testing standards, requirements, and record keeping
procedures is assessed independently by individuals other than those
performing the testing.

**Range finder:** Initial test performed to determine starting doses for
the main (definitive) test. The NRU assays test eight concentrations of
the test chemical or the positive control (PC) by diluting the stock
solution in log dilutions to cover a large concentration range.

**RC regression:** log (LD~50~) = 0.435 x log (IC~50~) + 0.625; for
estimating an LD~50~ value in mmol/kg (body weight) from an IC~50~ value
(in mM).

**Reduction alternative^2^:** A new or modified test method that reduces
the number of animals required.

**Reference substances:** Substances selected for use during the
research, development, prevalidation, and validation of a proposed test
method because their response in the *in vivo* reference test method or
the species of interest is known (see "reference test"). Reference
substances should represent the classes of chemicals for which the
proposed test method is expected to be used and cover the range of
expected responses (negative, weak to strong positive).

**Reference test method^2^:** The accepted *in vivo* test method used
for regulatory purposes to evaluate the potential of a test substance to
be hazardous to the species of interest.

**Refinement alternative^2^:** A new or modified test method that
refines procedures to lessen or eliminate pain or distress in animals or
enhances animal well-being.

**Registry of Cytotoxicity (RC):** Database that consists of *in vivo*
acute oral toxicity data (i.e., LD~50~ values) from rats and mice and
*in vitro* cytotoxicity data (i.e., IC~50~ values) from multiple cell
lines and cytotoxicity endpoints for 347 chemicals with known molecular
weights (Halle 1998). A regression model constructed from these data was
proposed by ZEBET, as a method to reduce animal use by identifying the
most appropriate starting doses for acute oral systemic toxicity tests

**Relevance^2^:** The extent to which a test method correctly predicts
or measures the biological effect of interest in humans or another
species of interest. Relevance incorporates consideration of the
"accuracy" or "concordance" of a test method.

**Reliability^2^:** A measure of the degree to which a test method can
be performed reproducibly within and among laboratories over time. It is
assessed by calculating intra- and inter-laboratory reproducibility and
intralaboratory repeatability.

**Replacement alternative^2^:** A new or modified test method that
replaces animals with nonanimal systems or one animal species with a
phylogenetically lower one (e.g., a mammal with an invertebrate).

**Reproducibility^2^:** The consistency of individual test results
obtained in a single laboratory (intralaboratory reproducibility) or in
different laboratories (interlaboratory reproducibility) using the same
protocol and test substances (see intra- and inter-laboratory
reproducibility).

**RTECS®:** Registry of Toxic Effects for Chemical Substances**.**
Compendium of data extracted from the open scientific literature. The
database includes toxicity data (e.g., acute toxicity) and specific
numeric toxicity values (e.g., LD~50~). Compiled by the U.S. National
Institute for Occupational Safety and Health (NIOSH) and now licensed to
MDL Information Systems, Inc.

**Sensitivity^2^:** The proportion of all positive substances that are
classified correctly as positive in a test method. It is a measure of
test method accuracy.

**Simulation modeling:** Computer simulation modeling of the acute
systemic toxicity assays to determine animal use. The simulation process
uses a simulated population of animals for testing, a reference endpoint
(i.e., "true" LD~50~ value), and its assumed log-normal distribution.
Morality is assumed to have a mean equal to the log of the true LD~50~.
The SD, which reflects the variability of the simulated population, is
the inverse of the slope of the dose-mortality curve. Due to a lack of
information for the real dose-mortality curve, the simulations assumed
slopes of 0.5, 0.8, 2, 4, and 8.3.

**Solubility:** The amount of a test substance that can be dissolved (or
thoroughly mixed with) culture medium or solvent. The solubility
protocol was based on a U.S. EPA guideline (EPA 1998) that involves
testing for solubility in a particular solvent, beginning at a
relatively high concentration and proceeding to successively lower
concentrations by adding more solvent as necessary for dissolution.
Testing stops when, upon visual observation, the procedure produces a
clear solution with no cloudiness or precipitate.

**Solvent control:** An untreated sample containing all components of a
test system, including the solvent that is processed with the test
substance-treated and other control samples to establish the baseline
response for the samples treated with the test substance dissolved in
the same solvent. When tested with a concurrent negative control, this
sample also demonstrates whether the solvent interacts with the test
system.

**Specificity^2^:** The proportion of all negative substances that are
classified correctly as negative in a test method. It is a measure of
test method accuracy.

**Spirit of GLP:** Guidance provided in the Statement of Work
specifically for the non GLP-compliant laboratory that participated in
the validation study. Based on the GLP standards referenced in the ECVAM
Workshop 37 Report (Cooper-Hannan 1999) and the OECD Principles of GLP
(OECD 1998). "*Laboratories that are non GLP-compliant shall adhere to
GLP principles and other method parameters. Documentation and
accountability shall be equal to GLP requirements. Laboratories must
make assurances that they are equal in performance criteria and that
there is parity amongst the laboratories."*

**TESS**: Toxic Exposure Surveillance System. A comprehensive poisoning
surveillance database maintained by the American Association of Poison
Control Centers (AAPCC).

**Test^2^:** The experimental system used; used interchangeably with
"test method" and "assay".

**Test method^2^:** A process or procedure used to obtain information on
the characteristics of a substance or agent. Toxicological test methods
generate information regarding the ability of a substance or agent to
produce a specified biological effect under specified conditions. Used
interchangeably with "test" and "assay". See also "validated test
method" and "reference test".

**Test method component:** Structural, functional, and procedural
elements of a test method that are used to develop the test method
protocol. These components include unique characteristics of the test
method, critical procedural details, and quality control measures.

**3T3:** BALB/c 3T3 clone A31 mouse fibroblasts developed in 1968 from
disaggregated 14- to 17-day-old BALB/c mouse embryos (American Type
Culture Collection \[ATCC\]; \# CCL-163).

**Tiered testing:** A testing strategy where all existing information on
a test substance is reviewed, in a specified order, before *in vivo*
testing.

**Toxicity** **underpredicted:** Actual LD~50~ value of a test substance
is lower than the predicted LD~50~ value.

**Toxicity** **overpredicted:** Actual LD~50~ value of a test substance
is higher than the predicted LD~50~ value.

**Transferability^2^:** The ability of a test method or procedure to be
accurately and reliably performed in different, competent laboratories.

**Up-and-Down Procedure (UDP):** An acute oral systemic toxicity test
method used to minimize the number of animals required to estimate the
acute oral toxicity of a chemical, estimate the LD~50~ and confidence
intervals (CI), and observe signs of toxicity. Single animals are tested
sequentially. Subsequent doses are based on the outcome of the previous
animal.

**Validated test method^2^:** An accepted test method for which
validation studies have been completed to determine the accuracy and
reliability of this method for a specific proposed use.

**Validation^2^:** The process by which the reliability and accuracy of
a procedure are established for a specific purpose.

**Vehicle control (VC):** The VC consists of appropriate cell culture
medium for the cells in the test (i.e., DMEM for 3T3 cells and
keratinocyte growth medium for the NHK cells). For chemicals dissolved
in DMSO, the VC consists of medium with the same amount of solvent as
that used in the test chemical concentrations that are applied to the
96-well test plate. The final DMSO concentration is ≤ 0.5 % (v/v) in the
VCs.

**Volatility:** Ability of a test chemical to evaporate. A general
indicator of volatility issues in the NRU test methods is the percent
difference in the mean OD values for the two VC columns on the test
plate. If the difference is greater than 15%, then chemical volatility
can be suspected, especially if the VC adjacent to the highest test
concentration had a significantly reduced OD value. Volatility may be an
issue for compounds with a specific gravity of less than 1.

**Weight of evidence (process):** The strengths and weaknesses of a
collection of information are used as the basis for a conclusion that
may not be evident from the individual data.

**Weight regressions:** Linear regressions with IC~50~ values in µg/mL
and LD~50~ values in mg/kg.

**ZEBET:** The German National Center for the Documentation and
Evaluation of Alternative Methods to Animal Experiments.

[^1]: The definitions in this Glossary are restricted to their uses with
    respect to *in vitro* cytotoxicity testing and the NRU test methods.

[^2]: Definition used by the Interagency Coordinating Committee on the
    Validation of Alternative Methods (ICCVAM 2003).