Methods

The x-axis of the activity profile represents the bioassays in phylogenetic sequence by endpoint, and the values on the y-axis represent the logarithmically transformed lowest effective doses (LED) and highest ineffective doses (HID) tested. The term ‘dose’, as used in this report, does not take into consideration length of treatment or exposure and may therefore be considered synonymous with concentration. In practice, the concentrations used in all the in-vitro tests were converted to μg/ml, and those for in-vivo tests were expressed as mg/kg bw. Because dose units are plotted on a log scale, differences in molecular weights of compounds do not, in most cases, greatly influence comparisons of their activity profiles. Conventions for dose conversions are given below.

Profile-line height (the magnitude of each bar) is a function of the LED or HID, which is associated with the characteristics of each individual test system — such as population size, cell-cycle kinetics and metabolic competence. Thus, the detection limit of each test system is different, and, across a given activity profile, responses will vary substantially. No attempt is made to adjust or relate responses in one test system to those of another.

Line heights are derived as follows: for negative test results, the highest dose tested without appreciable toxicity is defined as the HID. If there was evidence of extreme toxicity, the next highest dose is used. A single dose tested with a negative result is considered to be equivalent to the HID. Similarly, for positive results, the LED is recorded. If the original data were analysed statistically by the author, the dose recorded is that at which the response was significant (p < 0.05). If the available data were not analysed statistically, the dose required to produce an effect is estimated as follows: when a dose-related positive response is observed with two or more doses, the lower of the doses is taken as the LED; a single dose resulting in a positive response is considered to be equivalent to the LED.

In order to accommodate both the wide range of doses encountered and positive and negative responses on a continuous scale, doses are transformed logarithmically, so that effective (LED) and ineffective (HID) doses are represented by positive and negative numbers, respectively. The response, or logarithmic dose unit (LDU_ij), for a given test system i and chemical j is represented by the expressions

Fig. 1

Scale of log dose units used on the y-axis of activity profiles

In practice, an activity profile is computer generated. A data entry programme is used to store abstracted data from published reports. A sequential file (in ASCH) is created for each compound, and a record within that file consists of the name and Chemical Abstracts Service number of the compound, a three-letter code for the test system (see below), the qualitative test result (with and without an exogenous metabolic system), dose (LED or HID), citation number and additional source information. An abbreviated citation for each publication is stored in a segment of a record accessing both the test data file and the citation file. During processing of the data file, an average of the logarithmic values of the data subset is calculated, and the length of the profile line represents this average value. All dose values are plotted for each profile line, regardless of whether results are positive or negative. Results obtained in the absence of an exogenous metabolic system are indicated by a bar (−), and results obtained in the presence of an exogenous metabolic system are indicated by an upward-directed arrow (↑). When all results for a given assay are either positive or negative, the mean of the LDU values is plotted as a solid line; when conflicting data are reported for the same assay (i.e., both positive and negative results), the majority data are shown by a solid line and the minority data by a dashed line (drawn to the extreme conflicting response). In the few cases in which the numbers of positive and negative results are equal, the solid line is drawn in the positive direction and the maximal negative response is indicated with a dashed line.

Profile lines are identified by three-letter code words representing the commonly used tests. Code words for most of the test systems in current use in genetic toxicology were defined for the US Environmental Protection Agency's GENE-TOX Program (Waters, 1979; Waters & Auletta, 1981). For this publication, codes were redefined in a manner that should facilitate inclusion of additional tests in the future. If a test system is not defined precisely, a general code is used that best defines the category of the test. Naming conventions are described below.

Dose conversions for activity profiles

Doses are converted to µg/ml for in-vitro tests and to mg/kg bw per day for in-vivo experiments.

1. In-vitro test systems
   1. Weight/volume converts directly to µg/ml.
   2. Molar (M) concentration x molecular weight = mg/ml = 10³ µg/ml; mM concentration x molecular weight = µg/ml.
   3. Soluble solids expressed as % concentration are assumed to be in units of mass per volume (i.e., 1% = 0.01 g/ml = 10 000 µg/ml; also, 1 ppm = 1 µg/ml).
   4. Liquids and gases expressed as % concentration are assumed to be given in units of volume per volume. Liquids are converted to weight per volume using the density (D) of the solution (D = g/ml). If the bulk of the solution is water, then D = 1.0 g/ml. Gases are converted from volume to mass using the ideal gas law, PV = nRT. For exposure at 20–37°C at standard atmospheric pressure, 1% (v/v) = 0.4 µg/ml x molecular weight of the gas. Also, 1 ppm (v/v) = 4 × 10⁻⁵°g/ml x molecular weight.
   5. For microbial plate tests, concentrations reported as weight/plate are divided by top agar volume (if volume is not given, a 2-ml top agar is assumed). For spot tests, in which concentrations are reported as weight or weight/disc, a 1-ml volume is used as a rough approximation.
   6. Conversion of asbestos concentrations given in µg/cm² are based on the area (A) of the dish and the volume of medium per dish; i.e., for a 100-mm dish: A = πR² = π x (5cm)² = 78.5 cm². If the volume of medium is 10 ml, then 78.5 cm² = 10 ml and lcm² = 0.13 ml.
2. In-vitro systems using in-vivo activation
   • For the body fluid-urine (BF-) test, the concentration used is the dose (in mg.kg bw) of the compound administered to test animals or patients.
3. In-vivo test systems
   1. Doses are converted to mg/kg bw per day of exposure, assuming 100% absorption. Standard values are used for each sex and species of rodent, including body weight and average intake per day, as reported by Gold et al. (1984). For example, in a test using male mice fed 50 ppm of the agent in the diet, the standard food intake per day is 12% of body weight, and the conversion is dose = 50 ppm Õ 12% = 6 mg/kg bw per day.
      Standard values used for humans are: weight — males, 70 kg; females, 55 kg; surface area, 1.7 m²; inhalation rate, 201/min for light work, 301/min for mild exercise.
   2. When reported, the dose at the target site is used. For example, doses given in studies of lymphocytes of humans exposed in vivo are the measured blood concentrations in µg/ml.

Codes for test systems

For specific nonmammalian test systems, the first two letters of the three-symbol code word define the test organism (e.g., SA- for Salmonella typhimurium, EC- for Escherichia coli). In most cases, the first two letters accurately represent the scientific name of the organism. If the species is not known, the convention used is -S-. The third symbol may be used to define the tester strain (e.g., SA8 for S. typhimurium TA1538, ECW for E. coli WP2uvrA). When strain designation is not indicated, the third letter is used to define the specific genetic endpoint under investigation (e.g., —D for differential toxicity, —F for forward mutation, —G for gene conversion or genetic crossing-over, —N for aneuploidy, —R for reverse mutation, —U for unscheduled DNA synthesis). The third letter may also be used to define the general endpoint under investigation when a more complete definition is not possible or relevant (e.g., —M for mutation, —C for chromosomal aberration).

For mammalian test systems, the first letter of the three-letter code word defines the genetic endpoint under investigation: A— for aneuploidy, B— for binding, C— for chromosomal aberration, D— for DNA strand breaks, G— for gene mutation, I— for inhibition of intercellular communication, M— for micronucleus formation, R— for DNA repair, S— for sister chromatid exchange, T— for cell transformation and U— for unscheduled DNA synthesis.

For animal (i.e., nonhuman) test systems in vitro, when the cell type is not specified, the code letters -IA are used. For such assays in vivo, when the animal species is not specified, the code letters -VA are used. Commonly used animal species are identified by the third letter (e.g., —C for Chinese hamster, —M for mouse, —R for rat, —S for Syrian hamster).

For test systems using human cells in vitro, when the cell type is not specified, the code letters -IH are used. For assays on humans in vivo, when the cell type is not specified, the code letters -VH are used. Otherwise, the second letter specifies the cell type under investigation (e.g., -BH for bone marrow, -LH for lymphocytes).

Some other specific coding conventions used for mammalian systems are as follows: BF- for body fluids, HM- for host-mediated, —L for leucocytes or lymphocytes in vitro (-AL, animals; -HL, humans), -L- for leucocytes in vivo (-LA, animals; -LH, humans), —T for transformed cells.

Note that these are examples of major conventions used to define the assay code words. The alphabetized listing of codes must be examined to confirm a specific code word. As might be expected from the limitation to three symbols, some codes do not fit the naming conventions precisely. In a few cases, test systems are defined by first-letter code words, for example: MST, mouse spot test; SLP, mouse specific locus test, postspermatogonia; SLO, mouse specific locus test, other stages; DLM, dominant lethal test in mice; DLR, dominant lethal test in rats; MHT, mouse heritable translocation test.

The genetic activity profiles and listings that follow were prepared in collaboration with Environmental Health Research and Testing Inc. (EHRT) under contract to the US Environmental Protection Agency; EHRT also determined the doses used. The references cited in each genetic activity profile listing can be found in the list of references in the appropriate monograph.

References

• Garrett N.E., Stack H.F., Gross M.R., Waters M.D. An analysis of the spectra of genetic activity produced by known or suspected human carcinogens. Mutai. Res. 1984;134:89–111. [PubMed: 6504068]
• Gold L.S., Sawyer C.B., Magaw R., Backman G.M., de Veciana M., Levinson R., Hooper N.K., Havender W.R., Bernstein L., Peto R., Pike M.C., Ames B.N. A carcinogenic potency database of the standardized results of animal bioassays. Environ. Health Perspect. 1984;58:9–319. [PMC free article: PMC1569423] [PubMed: 6525996]
• Waters, M.D. (1979) The GENE-TOX pmgram. In: Hsie, AW, O'Neill, J.P. & McElheny, V.K., eds, Mammalian Cell Mutagenesis: The Maturation of Test Systems (Banbury Report 2), Cold Spring Harbor, NY, CHS Press, pp. 449–467.
• Waters M.D., Auletta A. The GENE-TOX program: genetic activity evaluation. J chem. Inf. comput. Sci. 1981;21:35–38. [PubMed: 7240352]