PART 795--PROVISIONAL TEST GUIDELINES Subpart A--[Reserved] Subpart B--Provisional Chemical Fate Guidelines Sec. 795.45 Inherent biogradability: Modified SCAS test for chemical substances that are water insoluble or water insoluble and volatile. 795.54 Anaerobic microbiological transformation rate data for chemicals in the subsurface environment. 795.70 Indirect photolysis screening test: Sunlight photolysis in waters containing dissolved humic substances. Subpart C--Provisional Environmental Effects Guidelines 795.120 Gammarid acute toxicity test. Subpart D--Provisional Health Effects Guidelines 795.223 Pharmacokinetic test. 795.225 Dermal pharmacokinetics of DGBE and DGBA. 795.228 Oral/dermal pharmacokinetics. 795.230 Oral and inhalation pharmacokinetic test. 795.231 Pharmacokinetics of isopropanal. 795.232 Inhalation and dermal pharmacokinetics of commercial hexane. 795.235 Toxicokinetic test. 795.250 Developmental neurotoxicity screen. 795.260 Subchronic oral toxicity test. 795.285 Morphologic transformation of cells in culture. Authority: 15 U.S.C. 2603. Subpart A--[Reserved] Subpart B--Provisional Chemical Fate Guidelines 795.45 Inherent biodegradability: Modified SCAS test for chemical substances that are water insoluble or water insoluble and volatile. (a) Introductory information--(1) Prerequisites. (i) Water solubility of the test chemical must be established. (ii) The organic carbon content of the test chemical must be established. (2) Guidance information. (i) Information on the relative proportions of the major components of the test chemical will be useful in interpreting the results obtained. (ii) Information on the toxicity of the chemical may be useful to the interpretation of low results and in the selection of appropriate test concentrations. (3) Standard documents. This Test Guideline has been based on the papers cited under paragraphs (d) (1) and (2) of this section. (b) Method--(1) Introduction, purpose, scope, relevance, application and limits of test--(i) The method. (A) The method is an adaptation of the Soap and Detergent Association Semi-Continuous Activated Sludge (SCAS) procedure for assessing the primary biodegradation of alkylbenzene sulphonate. The method involves exposure of the chemical to relatively high concentrations of microorganisms over a long time period (possibly several months). The viability of the microorganisms is maintained over this period by daily addition of a settled sewage feed. (B) Since the conditions provided by the test are highly favorable to the selection and/or adaptation of microorganisms capable of degrading the test chemical, the procedure may also be used to produce microbial inocula adapted to selected chemicals for use in other tests. The test is applicable to organic chemicals that are water insoluble or water insoluble and volatile and that are not inhibitory to bacteria at the test concentration. (ii) Reference chemicals. In some cases when investigating a new chemical, reference chemicals may be useful; however, specific reference chemicals cannot yet be recommended. Data on several chemicals used in interlaboratory tests are provided (see Table 1 in this paragraph) primarily so that calibration of the method may be performed from time to time and to permit comparison of results when another method is employed. Table 1--Examples of Results of SCAS Test on Various Chemicals Used in the OECD/EEC Interlaboratory Test 컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴 Percentage Ot- biodeg- Test chemical OT Oc radation (mg/l) (mg/l) bioelim- ination 컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴 4-Acetylaminobenzene sulphonate.... 17.2 2.0 85 Tetrapropylenebenzene sulphonate... 17.3 8.4 51.4 4-Nitrophenol...................... 16.9 0.8 95.3 Diethylene glycol.................. 16.5 0.2 98.8 Aniline............................ 16.9 1.7 95.9 Cyclopentane tetracarboxylate...... 17.9 3.2 81.1 컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴 Duration of test is 40 days, except 120 days for cyclopentane tetracarboxylate. (iii) Principle of the test method. (A) Activated sludge from a sewage treatment plant is placed in an aeration (SCAS) unit. The test chemical and settled domestic sewage are added, and the mixture is aerated for 23 hours. The aeration is then stopped, the sludge is allowed to settle, and the supernatant liquor is removed. The sludge remaining in the aeration chamber is then mixed with a further aliquot of test chemical and sewage and the cycle is repeated. (B) This method requires use of a chemical-specific analytical technique or 14C-labeled test chemical. The purpose of the method is to determine the fate of the test chemical in a conventional activated sludge treatment plant. To this end, a complete mass balance for the test chemical is established by quantifying parent chemical in settled effluent sludge solids (insoluble test chemicals whether volatile or not), effluent plus solids (insoluble test chemicals whether volatile or not), and off gases (volatile test chemicals only). The identification and quantification of degradation products in all phases are recommended, but not required. (iv) Quality criteria--(A) Reproducibility. When primary biodegradation is considered, very precise data are obtained for chemicals that are extensively degraded. The results reported in the reference under paragraph (d)(1) of this section suggest 95-percent confidence limits of less than ÷3 percent, and this includes interlaboratory tests. As would be expected, wider confidence limits are obtained for less biodegradable chemicals. (B) Possibility of standardization. Since the method uses a feed of settled sewage, absolute standardization is not possible unless this feed were replaced by synthetic sewage. However, since the method is designed to give an indication of the biodegradability potential of a chemical and is not a simulation test such standardization is unnecessary. (C) Possibility of automation. Automation of this method would be possible but would be expensive. As the method is not labor intensive, the exercise would offer few advantages. (2) Description of the test procedure--(i) Preparations. (A) The aeration units are cleaned and fixed in a suitable support. The air inlet tubes are connected to the supply manifold. A small laboratory-scale air compressor is used to aerate the units, and the air is presaturated with water to reduce evaporation losses from the units. (B) If the test chemical is volatile, exhaust gases from the aeration units shall be passed through a suitable trap (such as Amberlite XAD - 4, Rohm and Haas, Philadelphia, PA) to remove volatilized organics. (C) A sample of mixed liquor from an activated sludge plant treating predominantly domestic sewage is obtained. Approximately 150 milliliters (ml) of the mixed liquor are required for each aeration unit. (D) The organic carbon analyzer is calibrated using potassium hydrogen phthalate. (E) Stock solutions of the test chemicals are prepared: The concentration normally required is 400 milligrams per liter (mg/L) as organic carbon which gives a test chemical concentration of 20 mg/L carbon at the start of each aeration cycle if no biodegradation is occurring. (F) If the test chemical is insoluble in water at 400 mg/L it may be necessary to use ultrasound dispersion to obtain a uniform stable suspension. Alternatively, test chemical may be added directly to the aeration units. (G) The organic carbon content of the stock solutions is measured. (ii) Test conditions. A high concentration of aerobic microorganisms is used, and the effective detention period is 36 hours. The carbonaceous material in the sewage feed is oxidized extensively within 8 hours of the start of each aeration cycle. Thereafter, the sludge respires endogenously for the remainder of the aeration period, during which time the only available substrate is the test chemical unless this is also readily metabolized. These features, combined with daily reinoculation of the test when domestic sewage is used as the medium, provide highly favorable conditions for both adaptation and biodegradation. (iii) Performance of the test. (A) A sample of mixed liquor from a suitable activated sludge plant is obtained and aerated during transportation to the laboratory. Each aeration unit is filled with 150 ml of mixed liquor, and the aeration is started. After 23 hours, aeration is stopped, and the sludge is allowed to settle for 45 minutes. The tap is opened, and 100 ml of the supernatant liquor is withdrawn. A sample of settled domestic sewage is obtained immediately before use, and 100 ml are added to the sludge remaining in each aeration unit. Aeration is started anew. At this stage no test chemicals are added, and the units are fed daily with domestic sewage only until a clear supernatant liquor is obtained on settling. This usually takes up to 2 weeks, by which time the dissolved organic carbon in the supernatant liquor at the end of each aeration cycle should be less than 12 mg/L. (B) At the end of this period the individual settled sludges are mixed, and 50 ml of the resulting composite sludge are added to each unit. (C) One hundred ml of settled sewage are added to the control units, and 95 ml of settled sewage plus 5 ml of the appropriate test chemical stock solution or suspension (400 mg organic carbon/L) to the test units. If test chemical is added directly to aeration units, 100 ml of settled sewage is added, as in the control units. (D) Aeration is started again and continued for 23 hours. The sludge is then allowed to settle for 45 minutes and the supernatant drained off and analyzed for parent chemical. Before analysis the liquors are filtered through washed 0.45-micrometer membrane filters and certifuged. Temperature of the sample must not exceed 40 �C while it is in the centrifuge. (E) If the test chemical is insoluble or expected to sorb significantly to sludge solids, settled sludge is also collected by an appropriate means (such as centrifugation) and extracted to remove test chemical, and the extract is analyzed for parent chemical. (F) If the test chemical is volatile, traps for removing volatile organics from exhaust gases are also extracted and the extracts analyzed for parent chemical. (G) The fill and draw procedure under paragraphs (b)(2)(iii) (C) through (F) of this section is repeated daily throughout the test. (H) Before settling, it may be necessary to clean the walls of the units to prevent the accumulation of solids above the level of the liquid. A separate scraper or brush is used for each unit to prevent cross contamination. (I) The length of the test for chemicals showing little or no biodegradation is indeterminate, but experience suggests that this should be at least 12 weeks. (c) Data and reporting--(1) Treatment of the results. (i) The concentration of parent chemical in settled effluent sludge solids (insoluble test chemicals whether volatile or not), effluent plus solids (insoluble test chemicals whether volatile or not), and off-gases (volatile test chemicals only) is plotted versus time for the test units. As biodegradation is achieved the level of the test chemical will decrease and approach a steady state. Once the levels of the test chemical are found to be constant over three consecutive measurements, three further measurements are made. (ii) An example of the application of specific analytical technique to the SCAS test is discussed in the reference in paragraph (d)(2) of this section. (d) Literature references. For additional background information on this test guideline the following references should be consulted: (1) ``A Procedure and Standards for the Determination of the Biodegradability of Alkyl Benzene Sulphonate and Linear Alkylate Sulphonate'', Journal of the American Chemical Society, 42:986, 1965. (2) Games, L.M., King, J.E., and Larson, R.J. ``Fate and distribution of a quaternary ammonium surfactant octadecyltrimethylammonium chloride (OTAC), in wastewater treatment.'' Environmental Science and Technology, 16:483 - 488, 1982. (Approved by the Office of Management and Budget under control number 2070 - 0067) [52 FR 21026, June 4, 1987; 52 FR 32990, Sept. 1, 1987] 795.54 Anaerobic microbiological transformation rate data for chemicals in the subsurface environment. (a) Introduction. (1) This guideline describes laboratory methods for developing anaerobic microbiological transformation rate data for organic chemicals in subsurface materials. The method is based on a time-tiered approach. For chemicals that are degraded rapidly, only a portion (the 0, 4, and 8 week sampling periods, for example) of the test will have to be completed; however, for slowly degrading chemicals, the entire test may have to be performed (64 weeks). The data will be used to calculate degradation rate constants for each tested chemical over a range of environmental conditions. The rate constants obtained from testing will be integrated into algorithms to assess the fate of organic chemicals leaching into ground water from waste management facilities. (2) Anaerobic transformations are evaluated under methanogenic and sulfur-reducing conditions. Aerobic biodegradation was not included in the modeling analysis for two reasons: (i) Aerobic biodegradation would be limited by the concentration of oxygen in ground water. In the laboratory, oxygen would probably not be limiting, and the resulting degradation rates obtained would possibly be overestimations of actual subsurface degradation rates. (ii) Aerobic degradation would only occur at the leading edge of a contaminant plume where dispersion and other processes dilute the plume with oxygenated water, as stated in Wilson et al. (1985), in paragraph (d)(24) of this section. (3) The anaerobic transformation of chemicals in selected subsurface samples shall be estimated from subsurface microcosm studies using methods adapted from procedures recently reported by Wilson et al. (1986), in paragraph (d)(25) of this section. These procedures shall be used to determine the length of the adaptation period (time interval before detectable degradation of the chemical can be observed) and the half-life of the chemical following the adaptation period. Supporting laboratory methods shall be used to measure the levels of residual test chemical, intermediate degradation products, biomass, and other physical-chemical parameters. (b) Laboratory procedures--(1) Identification of subsurface sampling sites, collection of subsurface materials, and transportation and storage of subsurface materials.--(i) A minimum of six subsurface sampling sites shall be identified on the basis of two temperatures and three pH values. Three of the sites shall have annual average temperatures near 10 �C, and three of the sites shall have temperatures near 20 �C. These values are chosen to represent the high and low temperatures commonly observed in aquifers and are one standard deviation on either side of the mean temperature of 15 �C. Generally, low temperature sites are located in northern latitude areas of the United States, and high temperatures correspond to southern latitude areas. (ii) Acidic (pH 4.5 to 6.0), neutral (pH 6.5 to 7.5), and alkaline (pH 8.0 to 9.5) sites shall be selected for each temperature range. These ranges of pH values for ground waters are selected to estimate the effect of pH on microbial degradation capacity and to examine the effect of chemical form on the degradation of chemicals having dissociable hydrogen (i.e., degradation of the protonated and unprotonated forms of the chemical). Ground waters at all sites shall have dissolved-oxygen levels below 0.1 mg/L and sulfate concentrations below 10 mg/L. (iii) Samples of subsurface materials shall be collected in a manner that protects them from contamination from surface materials and maintains anaerobic conditions. An appropriate procedure has been reported by Wilson et al. (1983), in paragraph (d)(26) of this section. First, a bore hole is drilled to the desired depth with an auger. Then the auger is removed and the sample taken with a wireline piston core barrel, as reported by Zapico et al., 1987, in paragraph (d)(14) of this section. The core barrel is immediately transferred to an anerobic chamber, filled and continually purged with nitrogen gas, and all further manipulations are performed in the chamber. Using aseptic procedures, up to 5 centimeters (cm) of the core is extruded, then broken off to produce an uncontaminated face. A sterile paring device is then installed, and the middle 30 to 35 cm of the core is extruded, paring away the outer 1.0 cm of core material. As a result, the material that had been in contact with the core barrel, and thus might be contaminated with surface microorganisms, is discarded. Modifications of this technique can be used for samples obtained from deep coring devices when auger equipment is insufficient because of the depth of the aquifer. Subsurface material shall be stored under nitrogen gas and on ice and shall be used in microcosm studies within 7 days of collection. (iv) Ground waters will be collected from the bore hole used to collect subsurface materials. Ground waters will be pumped to the surface. The bore hole should be purged with argon before pumping begins. The pumping mechanism should be flushed with enough ground water to insure that a representative ground water sample is obtained. This flushing process generally requires a volume equal to 3 to 10 times the volume of water in the bore hole. Once flushing is complete, ground water samples should be collected, and stored under nitrogen and on ice for transport back to the laboratory. Ground waters shall be sterilized by filtration through 0.22 micrometer (�m) membranes on-site in a portable anaerobic chamber filled and continually purged with nitrogen gas. The sterile water shall be stored under nitrogen and on ice, and shall be used in microcosm studies within 7 days of collection. (v) Two samples shall be collected from each of the 6 sites. Each core sample shall be assayed for test chemical degradation and analyzed for biomass (heterotrophic, sulfate-reducing, and methanogenic) and physical-chemical parameters (pH, cation exchange capacity, total organic carbon, percent base saturation, percent silt, percent sand, percent clay, redox potential, percent ash-free dry weight). Each corresponding ground water sample will be analyzed for pH, dissolved oxygen, dissolved organic carbon, nutrients (sulfate, phosphate, nitrate), conductivity, and temperature. (2) Anaerobic Microcosm assay. (i) Microcosms shall consist of 160-milliliter (mL) serum bottles which have been filled completely with a slurry of subsurface material and ground water (20 grams equivalent dry wt (oven dry wt. 103 �C) solid to 80 mL ground water). One series of serum bottles shall be amended to a level of 200 mg/L sulfate (weight/volume added as sodium sulfate) to stimulate sulfate-reducing conditions. If the level of soluble sulfate falls below 50 mg/L at any sampling time, additional sulfate (200 mg/L, weight/volume) should be added to all remaining sulfate-amended microcosms. Soluble sulfate levels should be measured by the method of Watwood et al. (1986), in paragraph (d)(23) of this section. A second series shall be left unamended to simulate methanogenic conditions. All manipulations in preparing the microcosms shall be performed aseptically under strict anaerobic conditions, as described in Kaspar and Tiedje (1982) in paragraph (d)(10) of this section, or other equivalent methods, and all equipment in contact with the subsurface samples shall be sterilized. Sterile controls shall be prepared by autoclaving the samples for a minimum of 1 hour on each of 3 consecutive days. Test chemical amendments shall be prepared in sterile nitrogen-purged ground water. Sparingly soluble and volatile chemicals shall be added to sterile, nitrogen-purged ground water and then stirred overnight without a head space. (ii) The active and control microcosms shall be dosed with the test chemical and 0.0002 percent (w/v) Resazurin as a redox indicator, and then each unit shall be immediately sealed with a Teflon®-coated gray butyl rubber septum and crimp seal. As stated previously, all manipulations shall be performed under strict anaerobic conditions, as described in Kaspar and Tiedje (1982) in paragraph (d)(10) of this section, or other equivalent methods. The microcosms shall be stored in the dark at the original in-situ temperature. Active microcosms and control microcosms, randomly selected from the sulfate-amended series and the unamended series, shall be sacrificed and analyzed at 0, 4, 8, 16, 32, and 64 weeks for residual test chemical and the formation of degradation intermediates. Once the residual level of the chemical drops below 5 percent of the initial concentration, analysis of microcosms at subsequent time periods is not required. The active microcosms and control microcosms from both series, at weeks 0, 16, and 64 (or randomly selected from the remaining samples the week following 95 percent degradation of the chemical, if less than week 64) shall also be analyzed for heterotrophic, sulfate-reducing, and methanogenic bacteria. (iii) Three concentrations of each chemical tested shall be used. The test chemical concentrations should range between a low level of 30 times the health-based level and a level that equates to the chemical's solubility (or to a level that causes inhibition of the test chemical's degradation). (iv) Biomass measurements shall be made for heterotrophic, sulfate-reducing, and methanogenic bacteria. Biomass measurements have been included to insure comparability of results between samples of subsurface materials. Degradation rates derived from sediment samples having significantly high or low (student ``t'' test, 90 percent level) bacterial populations would not be considered in subsequent modeling efforts. Also, the ratio of sulfate-reducing organisms to methanogenic organisms would be used to determine if the dominant redox conditions were sulfate-reducing or methanogenic. Anaerobic techniques described by Kaspar and Tiedje (1982), cited in paragraph (d)(10) of this section, or other equivalent methods, shall be used. (v) Heterotrophic bacterial concentrations shall be measured by a modification of the procedure developed by Molongoski and Klug (1976) and Clark (1965), cited in paragraphs (d)(13) and (d)(6) of this section, respectively. A ten-mL sample taken from the center of the appropriate microcosm, which has been well mixed, shall be aseptically transferred to 100 mL of sterile dilution medium and agitated to suspend the organisms. Ten-mL samples shall then be transferred immediately from the center of the suspension to a 90-mL sterile dilution medium blank to give a 10−2 dilution; 10 mL shall be similarly transferred to another 90-mL of sterile dilution medium to obtain a dilution of 10−3. This process shall be repeated to give a dilution series through at least 10−7. Only the 10−1 dilution need be prepared from control samples. The dilution series can be modified to include dilutions of greater than 10−7, if necessary, and if sufficient sample is available. From the highest dilution, 0.1-mL portions shall be transferred to the surface of each of three dilute tryptone glucose extract agar plates. The sample shall be spread immediately over the surface of the plates; the process shall be repeated for lower dilutions. Dilute tryptone glucose agar plates shall be prepared by combining 24.0 g tryptone glucose extract agar in 1 liter of distilled water. The mixture shall be autoclaved, and 25 mL of the molten agar shall be transferred to petri plates. Agar plates should be stored in an anaerobic chamber for a minimum of 24 hours before use. The inoculated plates shall be incubated in plastic bags in the glove box, or, if necessary, removed and kept in anaerobic jars. After 14 days of incubation, the plates shall be examined and the total count per gram of dry sediment material shall be determined. If the plates from the most dilute sample show more than 300 colonies, the dilution series was inadequate. In this case, all of the plates shall be discarded, and the process shall be repeated with greater dilutions, as appropriate. (vi) Sulfate-reducing species shall be enumerated by the MPN (most probable number) technique as descibed in Pankhurst (1971) in paragraph (d)(15) of this section, or other equivalent method. The dilution series shall be prepared as described for heterotrophic bacteria. (vii) Methanogenic bacteria shall be enumerated by the MPN technique as described by Jones et al. (1982) in paragraph (d)(9) of this section, or by another equivalent method. The dilution series shall be prepared as described for heterotrophic bacteria. (3) Analytical measures of the loss of test chemical and intermediate degradation products. (i) The loss of test chemical shall be quantified by measuring the residual test chemical. The formation of degradation intermediates shall be quantified in microcosm assays for test chemicals that can potentially be transformed. Analysis for degradation intermediates shall be required when the level of test chemical has been reduced by more than 25 percent. Concentrations of the potential degradation products 1,2-, 1,3-, and 1,4-dichlorobenzene, and 1,2,4,5-tetrachlorobenzene shall be measured in the appropriate microcosms used to analyze the degradation of pentachlorobenzene. The concentration of the potential degradation product dibromomethane shall be measured in the appropriate microcosms used to analyze the degradation of bromoform. The potential degradation products methanethiol and chloromethane (methyl chloride) shall be measured in the appropriate microcosm used to analyze the degradation of trichloromethanethiol. The potential intermediate products 1,2-, 1,3-, and 1,4-dichlorobenzene shall be measured in the appropriate microcosm used to analyze the degradation of 1,2,4,5-tetrachlorobenzene. (ii) Measurements of test chemical and intermediate degradation products will require organic analytical techniques tailored to the specific test chemical and subsurface material being investigated. Several extraction and purge-trap techniques are available for the recovery of residual test chemicals and degradative intermediates from subsurface materials. Unique analytical procedures would have to be developed or modified for each test chemical and sediment. The following represent examples of such techniques: (A) Soxlet extraction as described in Anderson et al. (1985), Bossart et al. (1984), Eiceman et al. (1986), Grimalt et al. (1984), and Kjolholt (1985) in paragraphs (d) (2), (3), (7), (8), and (11) of this section, respectively. (B) Shake flask method as described in Brunner et al. (1985), and Russel and McDuffie (1983) in paragraphs (d) (4) and (16) of this section, respectively. (C) Sonification as described in Schellenberg et al. (1984) in paragraph (d)(17) of this section. (D) Homogenization as described in Fowlie and Sulman (1986), Lopez-Avila et al. (1983), Sims et al. (1982), Stott and Tabatabai (1985), and U.S. EPA (1982) in paragraphs (d) (5), (12), (18), (19), and (22) of this section, respectively. (E) Purge-trap techniques have been described by Wilson et al. (1986) in paragraph (d)(24) of this section. (iii) These procedures can be readily coupled to gas chromatography (GC) and high-pressure liquid chromatography (HPLC) procedures to quantify the chemicals of interest. Whatever analytical procedure is selected shall follow Good Laboratory Practice Standards of 40 CFR part 792. (4) Characterization of subsurface materials and ground waters. (i) Subsurface materials shall be classified, described, and characterized as to soil type and physical and chemical properties using standard procedures as described by the Soil Conservation Service (U.S. Department of Agriculture, 1972 and 1975) in paragraphs (d) (20) and (21) of this section, or other equivalent methods. Ten parameters will be measured as follows: (A) Total organic carbon (TOC). (B) pH. (C) Cation exchange capacity. (D) Percent base saturation. (E) Percent silt. (F) Percent sand. (G) Percent clay. (H) Redox potential. (I) Percent ash-free dry weight. (J) Texture. (ii) Ground water shall be characterized for the following, by standard water and wastewater methods described by the American Public Health Association (1985) in paragraph (d)(1) of this section, or other equivalent methods: (A) pH. (B) Dissolved oxygen. (C) Dissolved organic carbon. (D) Nutrients including sulfate, phosphate, and nitrate. (E) Conductivity. (F) Temperature. (iii) The properties of pH, dissolved oxygen, and temperature shall be measured at the site of collection. All other properties shall be measured in the laboratory. (c) Data to be reported to the Agency. Data shall be reported for the two subsurface samples and corresponding ground waters taken from the six different sampling sites. (1) The following shall be reported for subsurface sediment samples: (i) Levels of residual test chemicals (mg/gm/dry wt) quantified in each of the randomly selected replicate microcosm and sterile controls at the specific time periods identified under the anaerobic microcosm assay. (ii) Numbers of heterotrophic, sulfate-reducing, and methanogenic bacteria (colony forming units (CFU) or most probable number units (MPNU) per gm dry wt) enumerated in each replicate microcosm and sterile controls at the specific time periods identified under the anaerobic microcosm assay. (iii) Levels of persistent degradation intermediates identified in microcosm and sterile controls at the specific time periods identified under the anaerobic microcosm assay. (iv) Measured values for pH, cation exchange capacity (meg/100 gm dry wt), percent base saturation, percent silt (percent dry wt), percent sand (percent dry wt), percent clay (percent dry wt), redox potential (Eh, Standard Hydrogen Electrode), percent ash free dry weight (percent dry wt), and a description of texture. (2) For ground water samples, the analysis report shall provide measured values for: (i) pH. (ii) Dissolved oxygen (mg/L). (iii) Dissolved organic carbon (mg/L). (iv) Nutrients including sulfate (mg/L), phosphate (mg/L), and nitrate (mg/L). (v) Conductivity (umho, 25 �C). (vi) Temperature (�C). (d) References. For additional background information cited in this protocol, the following references should be consulted: (1) American Public Health Association, American Water Works Association, and Water Pollution Control Federation. ``Standard methods for the examination of water and wastewater,'' 16th ed., A.E. Greenberg, R.R. Trussel, and L.C. Clesceri (eds.), American Public Health Association, Washington, DC (1985). (2) Anderson, J.W., G.H. Herman, D.R. Theilen, and A.F. Weston. ``Method verification for determination of tetrachlorodibenzodioxine in soil.'' Chemosphere 14:1115 - 1126 (1985). (3) Bossart, I., W.M. Kachal, and R. Bartha. ``Fate of hydrocarbons during oil sludge disposal in soil.'' Applied and Environmental Microbiology 47:763 - 767 (1984). (4) Brunner, W., F.H. Sutherland, and D.D. Focht. ``Enhanced biodegradation of polychlorinated biphenyls in soil by analog enrichment and bacterial inoculation.'' Journal of Environmental Quality 14:324 - 328 (1985). (5) Fowlie, P.J.A., and T.L. Bulman. ``Extraction of anthracene and benzo(a)-pyrene from soil.'' Analytical Chemistry 58:721 - 723 (1986). (6) Clark, F.E. ``Agar-plate method for total microbial count,'' p. 1460 - 1466. In: C.A. Black (ed.), ``Methods of soil analysis. Part 2. Chemical and Microbiological Properties.'' American Society of Agronomy, Inc., Madison WI (1965). (7) Eiceman, G.A., B. Davani, and J. Ingram. ``Depth profiles for hydrocarbons and polycyclic aromatic hydrocarbons in soil beneath waste disposal pits from natural gas production.'' Environmental Science and Technology. 20:508 - 514 (1986). (8) Grimalt, J., C. Marfil, and J. Albaiges. ``Analysis of hydrocarbons in aquatic sediments.'' International Journal of Environmental Analytical Chemistry 18:183 - 194 (1984). (9) Jones, J.G., B.M. Simon, and S. Gardener. ``Factors affecting methanogenesis and associated anaerobic processes in the sediments of a stratified eutrophic lake.'' Journal of General Microbiology 128:1 - 11 (1982). (10) Kaspar, H.F., and J.M. Tiedje. ``Anaerobic bacteria and processes,'' p. 989 - 1009. In: A.L. Page (ed.), ``Methods of Soil Analysis. Part 2. Chemical and Microbiological Properties.'' American Society of Agronomy, Madison, WI (1982). (11) Kjolholt, J. ``Determination of trace amounts of organophosphorous pesticides and related compounds in soils and sediments using capillary gas chromatography and a nitrogen-phosphorous detector.'' Journal of Chromatography 325:231 - 238 (1985). (12) Lopez-Avila, V., R. Northcutt, J. Onstot, M. Wickham, and S. Billets. ``Determination of 51 priority organic compounds after extraction from standard reference materials.'' Analytical Chemistry 55:881 - 889 (1983). (13) Molongoski, J.J., and M.J. Klug. ``Characterization of anaerobic heterotrophic bacteria isolated from freshwater lake sediments.'' Applied Environmental Microbiology 31:83 - 90 (1976). (14) Zapico, M.M., S. Vales, and J.S. Cherry. ``A wireline piston core barrel for sampling cohesionless sand and gravel below the water table.'' Ground Water Monitoring Review. Summer, Vol. 7, No. 3:74 - 82 (1987). (15) Pankhurst, E.S. ``The isolation and enumeration of sulphate-reducing bacteria,'' p. 223 - 240. In: D.A. Shapton and R.G. Board (eds.), ``Isolation of Anaerobes.'' Academic Press, Inc., New York (1971). (16) Russell, D.J., and B. McDuffie. ``Analysis for phthalate esters in environmental samples: Separation from PCBs and pesticides using dural column chromatography.'' International Journal of Environmental Analytical Chemistry 15:165 - 183 (1983). (17) Schellenberg, K., C.L. Leuenberger, and R.P. Schwarzenback. ``Sorption of chlorinated phenols by natural sediments and aquifer materials.'' Environmental Science and Technology. 18:652 - 657 (1984). (18) Sims, R.C. ``Land treatment of polynuclear wastes.'' Ph.D. dissertation. North Carolina State University, Raleigh, NC (1982). (19) Stott, D.E., and M.A. Tabatabai. ``Identification of phospholipids in soils and sewage sludges by high-performance liquid chromatography.'' Journal of Environmental Quality. 14:107 - 110 (1985). (20) United States Department of Agriculture. ``Soil survey laboratory methods and procedures for collecting soil samples.'' Soil Survey Investigations Report No. 1. Soil Conservation Service. Soil Survey Investigation (1972). (21) United States Department of Agriculture. ``Soil taxonomy: a basic system of soil classification for making and interpreting soil surveys.'' Agricultural Handbook 436. Soil Conservation Service (1975). (22) U.S. Environmental Protection Agency. ``Test Methods for Evaluating Solid Wastes: Physical/Chemical Methods.'' Second Edition. SW - 846. U.S. Environmental Protection Agency, Washington, DC (1982). (23) Watwood, M.E., J.W. Fitzgerald, and J.R. Gosz. Sulfur processing in forest soil and litter along an elevational and vegetative gradient. Canadian Journal of Forest Resources. 16:689 - 695 (1986). (24) Wilson, J.T., J.F. McNabb, J.W. Cochran, T.H. Wang, M.B. Tomson, and P.B. Bedient. ``Influence of microbial adaptation of the fate of organic pollutants in ground water.'' Environmental Toxicology and Chemistry. 4:721 - 726 (1985). (25) Wilson, B.H., G.B. Smith, and J.F. Rees. ``Biotransformation of selected alkylbenzenes and halogenated aliphatic hydrocarbons in methanogenic aquifer material: A microcosm study.'' Environmental Science and Technology. 20:997 - 1002 (1986). (26) Wilson, J.T., J.F. McNabb, D.L. Balwill, and W.C. Ghiorse. ``Enumeration and characterization of bacteria indigenous to a shallow water-table aquifer.'' Groundwater 21:134 - 142 (1983). [53 FR 22320, June 15, 1988] 795.70 Indirect photolysis screening test: Sunlight photolysis in waters containing dissolved humic substances. (a) Introduction. (1) Chemicals dissolved in natural waters are subject to two types of photoreaction. In the first case, the chemical of interest absorbs sunlight directly and is transformed to products when unstable excited states of the molecule decompose. In the second case, reaction of dissolved chemical is the result of chemical or electronic excitation transfer from light-absorbing humic species in the natural water. In contrast to direct photolysis, this photoreaction is governed initially by the spectroscopic properties of the natural water.In general, both indirect and direct processes can proceed simultaneously. Under favorable conditions the measurement of a photoreaction rate constant in sunlight (KpE) in a natural water body will yield a net value that is the sum of two first-order reaction rate constants for the direct (kDE) and indirect (kIE) pathways which can be expressed by the relationship Equation 1 kpE=kDE+kIE. This relationship is obtained when the reaction volume is optically thin so that a negligible fraction of the incident light is absorbed and is sufficiently dilute in test chemical; thus the direct and indirect photoreaction processes become first-order. (3) In pure water only, direct photoreaction is possible, although hydrolysis, biotransformation, sorption, and volatilization also can decrease the concentraton of a test chemical. By measuring kpE in a natural water and kDE in pure water, kIE can be calculated. (4) Two protocols have been written that measure kDE in sunlight or predict kDE in sunlight from laboratory measurements with monochromatic light (USEPA (1984) under paragraph (f)(14) and (15) of this section; Mill et al. (1981) under paragraph (f)(9) of this section; Mill et al. (1982) under paragraph (f)(10) of this section; Mill et al. (1983) under paragraphs (f)(11) of this section). As a preface to the use of the present protocol, it is not necessary to know kDE; it will be determined under conditions that definitively establish whether kIE is significant with respect to kDE. (5) This protocol provides a cost effective test method for measuring kIE for test chemicals in a natural water (synthetic humic water, SHW) derived from commercial humic material. It describes the preparation and standardization of SHW. To implement the method, a test chemical is exposed to sunlight in round tubes containing SHW and tubes containing pure water for defined periods of time based on a screening test. (6) To correct for variations in solar irradiance during the reaction period, an actinometer is simultaneously insolated. From these data, an indirect photoreaction rate constant is calculated that is applicable to clear-sky, near-surface, conditions in fresh water bodies. (7) In contrast to kDE, which, once measured, can be calculated for different seasons and latitudes, kIE only applies to the season and latitude for which it is determined. This condition exists because the solar action spectrum for indirect photoreaction in humic-containing waters is not generally known and would be expected to change for different test chemicals. For this reason, kpE, which contains kIE, is likewise valid only for the experimental data and latitude. (8) The value of kpE represents an atypical quantity because kIE will change somewhat from water body to water body as the amount and quality of dissolved aquatic humic substances change. Studies have shown, however, that for optically-matched natural waters, these differences are usually within a factor of two (Zepp et al. (1981) under paragraph (f)(17) of this section). (9) This protocol consists of three separate phases that should be completed in the following order: In Phase 1, SHW is prepared and adjusted; in Phase 2, the test chemical is irradiated in SHW and pure water (PW) to obtain approximate sunlight photoreaction rate constants and to determine whether direct and indirect photoprocesses are important; in Phase 3, the test chemical is again irradiated in PW and SHW. To correct for photobleaching of SHW and also solar irradiance variations, tubes containing SHW and actinometer solutions are exposed simultaneously. From these data kpE is calculated that is the sum of kIE and kDE (Equation 1) (Winterle and Mill (1985) under paragraph (f)(12) of this section). (b) Phase 1--Preparation and standardization of synthetic natural water--(1) Approach. (i) Recent studies have demonstrated that natural waters can promote the indirect (or sensitized) photoreaction of dissolved organic chemicals. This reactivity is imparted by dissolved organic material (DOM) in the form of humic substances. These materials absorb sunlight and produce reactive intermediates that include singlet oxygen (102) (Zepp et al. (1977) under paragraph (f)(20) of this section, Zepp et al. (1981) under paragraph (f)(17) of this section, Zepp et al. (1981) under paragraph (f)(18) of this section, Wolff et al. (1981) under paragraph (f)(16) of this section, Haag et al. (1984) under paragraph (f)(6) of this section, Haag et al. (1984) under paragraph (f)(7) of this section); peroxy radicals (RO2&tbull;) (Mill et al. (1981) under paragraph (f)(9) of this section; Mill et al. (1983) under paragraph (f)(8) of this section); hydroxyl radicals (HO&tbull;) (Mill et al. (1981) under paragraph (f)(9) of this section, Draper and Crosby (1981, 1984) under paragraphs (f)(3) and (4) of this section); superoxide anion (02−&tbull;) and hydroperoxy radicals (HO&tbull;). (Cooper and Zika (1983) under paragraph (f)(1) of this section, Draper and Crosby (1983) under paragraph (f)(2) of this section); and triplet excited states of the humic substances (Zepp et al. (1981) under paragraph (f)(17) of this section, Zepp et al. (1985) under paragraph (f)(21) of this section). Synthetic humic waters, prepared by extracting commercial humic or fulvic materials with water, photoreact similarly to natural waters when optically matched (Zepp et al. (1981) under paragraphs (f)(17) and (18) of this section). (ii) The indirect photoreactivity of a chemical in a natural water will depend on its response to these reactive intermediates, and possibly others yet unknown, as well as the ability of the water to generate such species. This latter feature will vary from water-to-water in an unpredictable way, judged by the complexity of the situation. (iii) The approach to standardizing a test for indirect photoreactivity is to use a synthetic humic water (SHW) prepared by water-extracting commercial humic material. This material is inexpensive, and available to any laboratory, in contrast to a specific natural water. The SHW can be diluted to a dissolved organic carbon (DOC) content and uv-visible absorbance typical of most surface fresh waters. (iv) In recent studies it has been found that the reactivity of SHW mixtures depends on pH, and also the history of sunlight exposure (Mill et al. (1983) under paragraph (f)(11) of this section). The SHW solutions initially photobleach with a time-dependent rate constant. As such, an SHW test system has been designed that is buffered to maintain pH and is pre-aged in sunlight to produce, subsequently, a predictable bleaching behavior. (v) The purpose of Phase 1 is to prepare, pre-age, and dilute SHW to a standard mixture under defined, reproducible conditions. (2) Procedure. (i) Twenty grams of Aldrich humic acid are added to a clean 2-liter Pyrex Erlenmeyer flask. The flask is filled with 2 liters of 0.1 percent NaOH solution. A stir bar is added to the flask, the flask is capped, and the solution is stirred for 1 hour at room temperature. At the end of this time the dark brown supernatant is decanted off and either filtered through coarse filter paper or centrifuged and then filtered through 0.4 )m microfilter. The pH is adjusted to 7.0 with dilute H2SO4 and filter sterilized through a 0.2 )m filter into a rigorously cleaned 2-liter Erlenmeyer flask. This mixture contains roughly 60 ppm DOC and the absorbance (in a 1 cm path length cell) is approximately 1.7 at 313 nm and 0.7 at 370 nm. (ii) Pre-aging is accomplished by exposing the concentrated solution in the 2-liter flask to direct sunlight for 4 days in early spring or late fall; 3 days in late spring, summer, or early fall. At this time the absorbance of the solution is measured at 370 nm, and a dilution factor is calculated to decrease the absorbance to 0.50 in a 1 cm path length cell. If necessary, the pH is re-adjusted to 7.0. Finally, the mixture is brought to exact dilution with a precalculated volume of reagent-grade water to give a final absorbance of 0.500 in a 1-cm path length cell at 370 nm. It is tightly capped and refrigerated. (iii) This mixture is SHW stock solution. Before use it is diluted 10-fold with 0.010 M phosphate buffer to produce a pH 7.0 mixture with an absorbance of 5.00 x 10−2 at 370 nm, and a dissolved organic carbon of about 5 ppm. Such values are characteristic of many surface fresh waters. (3) Rationale. The foregoing procedure is designed to produce a standard humic-containing solution that is pH controlled, and sufficiently aged that its photobleaching first-order rate constant is not time dependent. It has been demonstrated that after 7 days of winter sunlight exposure, SHW solutions photobleached with a nearly constant rate constant (Mill et al. (1983) under paragraph (f)(11) of this section). (c) Phase 2--Screening test--(1) Introduction and purpose. (i) Phase 2 measurements provide approximate solar photolysis rate constants and half-lives of test chemicals in PW and SHW. If the photoreaction rate in SHW is significantly larger than in PW (factor of > 2X) then the test chemical is subject to indirect photoreaction and Phase 3 is necessary. Phase 2 data are needed for more accurate Phase 3 measurements, which require parallel solar irradiation of actinometer and test chemical solutions. The actinometer composition is adjusted according to the results of Phase 2 for each chemical, to equalize as much as possible photoreaction rate constants of chemical in SHW and actinometer. (ii) In Phase 2, sunlight photoreaction rate constants are measured in round tubes containing SHW and then mathematically corrected to a flat water surface geometry. These rate constants are not corrected to clear-sky conditions. (2) Procedure. (i) Solutions of test chemicals should be prepared using sterile, air-saturated, 0.010 M, pH 7.0 phosphate buffer and reagent-grade (or purer) chemicals.The water should be ASTM Type IIA, or an equivalent grade. Reaction mixtures should be prepared with chemicals at concentrations at less than one-half their solubility in pure water and at concentrations such that, at any wavelengths above 290 nm, the absorbance in a standard quartz sample cell with a 1-cm path length is less than 0.05. If the chemicals are too insoluble in water to permit reasonable handling or analytical procedures, 1-volume percent acetonitrile may be added to the buffer as a cosolvent. (ii) This solution should be mixed 9.00:1.00 by volume with PW or SHW stock solution to provide working solutions. In the case of SHW, it gives a ten-fold dilution of SHW stock solution. Six mL aliquots of each working solution should then be transferred to separate 12 x 100 mm quartz tubes with screw tops and tightly sealed with Mininert valves.Mininert Teflon sampling vials are available from Alltech Associates, Inc., 202 Campus Dr., Arlington Heights, IL 60004. Twenty four tubes are required for each chemical solution (12 samples and 12 dark controls), to give a total of 48 tubes. (iii) The sample tubes are mounted in a photolysis rack with the tops facing geographically north and inclined 30� from the horizontal. The rack should be placed outdoors over a black background in a location free of shadows and excessive reflection. (iv) Reaction progress should be measured with an analytical technique that provides a precision of at least ÷5 percent. High pressure liquid chromatography (HPLC) or gas chromatograph (GC) have proven to be the most general and precise analytical techniques. (v) Sample and control solution concentrations are calculated by averaging analytical measurements for each solution. Control solutions should be analyzed at least twice at zero time and at other times to determine whether any loss of chemical in controls or samples has occurred by some adventitious process during the experiment. (vi) Whenever possible the following procedures should be completed in clear, warm, weather so that solutions will photolyze more quickly and not freeze. (A) Starting at noon on day zero, expose to sunlight 24 sample tubes mounted on the rack described above. Tape 24 foil-wrapped controls to the bottom of the rack. (B) Analyze two sample tubes and two unexposed controls in PW and SHW for chemical at 24 hours. Calculate the round tube photolysis rate constants (kp)SHW and (kp)W if the percent conversions are J 20 percent but F 80 percent. The rate constants (kp)SHW and (kp)W are calculated, respectively, from Equations 2 and 3: Equation 2 (kp)SHW=(1/t)Pn(Co/Ct)SHW (in d−1) Equation 3 (kp)W=(1/t)Pn(Co/Ct)W (in d−1), where the subscript identifies a reaction in SHW or PW; t is the photolysis time in calendar days; Co is the initial molar concentration; and Ct is the molar concentration in the irradiated tube at t. In this case t=1 day. (C) If less than 20 percent conversion occurs in SHW in 1 day, repeat the procedure for SHW and PW at 2 days, 4 days, 8 days, or 16 days, or until 20 percent conversion is reached. Do not extend the experiment past 16 days. If less than 20 percent photoreaction occurs in SHW at the end of 16 days the chemical is ``photoinert''. Phase 3 is not applicable. (D) If more than 80 percent photoreaction occurs at the end of day 1 in SHW, repeat the experiment with eight each of the remaining foil-wrapped PW and SHW controls. Divide these sets into four sample tubes each, leaving four foil-wrapped controls taped to the bottom of the rack. (1) Expose tubes of chemical in SHW and PW to sunlight starting at 0900 hours and remove one tube and one control at 1, 2, 4, and 8 hours. Analyze all tubes the next day. (2) Extimate (kp)SHW for the first tube in which photoreaction is J 20 percent but F 80 percent. If more than 80 percent conversion occurs in the first SHW tube, report: ``The half-life is less than one hour'' and end all testing. The chemical is ``photolabile.'' Phase 3 is not applicable. (3) The rate constants (kp)SHW and (kp)W are calculated from equations 2 and 3 but the time of irradiation must be adjusted to reflect the fact that day-averaged rate constants are approximately one-third of rate constants averaged over only 8 daylight hours. For 1 hour of insolation enter t=0.125 day into equation 2. For reaction times of 2, 4, and 8 hours enter 0.25, 0.50 and 1.0 days, respectively. Proceed to Phase 3 testing. (4) Once (kp)SHW and (kp)W are measured, determine the ratio R from equation 4: Equation 4 R=(kp)SHW/(kp)W. The coefficient R, defined by Equation 4, is equal to [(kI+kD)/kD]. If R is in the range 0 to 1, the photoreaction is inhibited by the synthetic humic water and Phase 3 does not apply. If R is in the range 1 to 2, the test chemical is marginally susceptable to indirect photolysis. In this case, Phase 3 studies are optional. If R is greater than 2, Phase 3 measurements are necessary to measure kpE and to evaluate kIE. (vii) Since the rate of photolysis in tubes is faster than the rate in natural water bodies, values of near-surface photolysis rate constants in natural and pure water bodies, kpE and kDE, respectively, can be obtained from (kp)SHW and (kp)W from Equations 5 and 6: Equation 5 kpE=0.45(kp)SHW Equation 6 kDE=0.45(kp)W. The factor 0.45 is an approximate geometric correction for scattered light in tubes versus horizontal surfaces. A rough value of kIE, the rate constant for indirect photolysis in natural waters or SHW, can be estimated from the difference between kpE and kDE using Equation 7: Equation 7 kIE=kpE훛DE. (3) Criteria for Phase 2. (i) If no loss of chemical is found in dark control solutions compared with the analysis in tubes at zero time (within experimental error), any loss of chemical in sunlight is assumed to be due to photolysis, and the procedure provides a valid estimate of kpE and kDE. Any loss of chemical in the dark-control solutions may indicate the intervention of some other loss process such as hydrolysis, microbial degradation, or volatilization. In this case, more detailed experiments are needed to trace the problem and if possible eliminate or minimize the source of loss. (ii) Rate constants determined by the Phase 2 protocol depend upon latitude, season, and weather conditions. Note that (kp)SHW and kD values apply to round tubes and kpE and kDE values apply to a natural water body. Because both (kp)SHW and kD are measured under the same conditions the ratio ((kp)SHW/kD) is a valid measure of the susceptibility of a chemical to indirect photolysis. However, since SHW is subject to photobleaching, (kp)SHW will decrease with time because the indirect rate will diminish. Therefore, R >2 is considered to be a conservative limit because (kp)SHW will become systematically smaller with time. (4) Rationale. The Phase 2 protocol is a simple procedure for evaluating direct and indirect sunlight photolysis rate constants of a chemical at a specific time of year and latitude. It provides a rough rate constant for the chemical in SHW that is necessary for Phase 3 testing. By comparison with the direct photoreaction rate constant, it can be seen whether the chemical is subject to indirect photoreaction and whether Phase 3 tests are necessary. (5) Scope and limitations. (i) Phase 2 testing separates test chemicals into three convenient categories: ``Photolabile'', ``photoinert'', and those chemicals having sunlight half-lives in round tubes in the range of 1 hour to 50 days. Chemicals in the first two categories fall outside the practical limits of the test, and cannot be used in Phase 3. All other chemicals are suitable for Phase 3 testing. (ii) The test procedure is simple and inexpensive, but does require that the chemical dissolve in water at sufficient concentrations to be measured by some analytical technique but not have appreciable absorbance in the range 290 to 825 nm. Phase 2 tests should be done during a clear-sky period to obtain the best results. Testing will be less accurate for chemicals with half-lives of less than 1 day because dramatic fluctuations in sunlight intensity can arise from transient weather conditions and the difficulty of assigning equivalent reaction times. Normal diurnal variations also affect the photolysis rate constant. Phase 3 tests should be started as soon as possible after the Phase 2 tests to ensure that the (kp)SHW estimate remains valid. (6) Illustrative Example. (i) Chemical A was dissolved in 0.010 M pH 7.0 buffer. The solution was filtered through a 0.2 )m filter, air saturated, and analyzed. It contained 1.7 x 10−5 M A, five-fold less than its water solubility of 8.5 x 10−5 M at 25�C. A uv spectrum (1-cm path length) versus buffer blank showed no absorbance greater than 0.05 in the wavelength interval 290 to 825 nm, a condition required for the Phase 2 protocol. The 180 mL mixture was diluted by the addition of 20 mL of SHW stock solution. (ii) The SHW solution of A was photolyzed in sealed quartz tubes (12 x 100 mm) in the fall season starting on October 1. At the end of 1 and 2 days, respectively, the concentration of A was found to be 1.13 x 10−5 M and 0.92 x 10−5 M compared to unchanged dark controls (1.53 x 10−5 M). (iii) The tube photolysis rate constant of chemical A was calculated from Equation 2 under paragraph (c)(2)(vi)(B) of this section. The first time point at day 1 was used because the fraction of A remaining was in the range 20 to 80 percent: (kp)SHW=(1/1d)Pn(1.53 x 10−5/1.13 x 10−5) (kp)SHW=0.30 d−1. (iv) From this value, kpE was found to be 0.14 d�1 using equation 5 under paragraph (c)(2)(vii) of this section: kpE=0.45(0.30 d−1)=0.14d−1. (v) From measurements in pure water, kD for chemical A was found to be 0.085 d−1. Because the ratio of (kp)SHW/kD(=3.5) is greater than 2, Phase 3 experiments were started. (d) Phase 3--Indirect photoreaction with actinometer: Calculation of kIE and kpE--(1) Introduction and purpose. (i) The purpose of Phase 3 is to measure kIo, the indirect photolysis rate constant in tubes, and then to calculate kpE for the test chemical in a natural water. If the approximate (kp)SHW determined in Phase 2 is not significantly greater than kD measured for the experiment date of Phase 2, then Phase 3 is unnecessary because the test chemical is not subject to indirect photoreaction. (ii) In the case (kp)SHW is significantly larger than kD, Phase 3 is necessary. The rate constant (kp)SHW is used to choose an actinometer composition that matches the actinometer rate to the test chemical rate. Test chemical solutions in SHW and in pure water buffer are then irradiated in sunlight in parallel with actinometer solutions, all in tubes. (iii) The actinometer used is the p-nitroacetophenone-pyridine (PNAP/PYR) system developed by Dulin and Mill (1982) under paragraph (f)(5) of this section and is used in two EPA test guidelines (USEPA (1984) under paragraphs (f) (14) and (15) of this section). By varying the pyridine concentration, the PNAP photolysis half-life can be adjusted over a range of several hours to several weeks. The starting PNAP concentration is held constant. (iv) SHW is subject to photobleaching that decreases its ability to promote indirect photolysis based on its ability to absorb sunlight. This effect will be significant when the test period exceeds a few days. To correct for photobleaching, tubes containing SHW are irradiated in action to the other tubes above. (v) At any time, the loss of test chemical is given by Equation 8 assuming actinometric correction to constant light flux: Equation 8 �(d[C]/dt)=kI[C]+kD[C]. (vi) The indirect photolysis rate constant, kI, is actually time dependent because SHW photobleaches; the rate constant kI, after pre-aging, obeys the formula: Equation 9 kI=kIo exp(훛t), in which kIo is the initial indirect photoreaction rate constant and k is the SHW photobleaching rate constant. After substituting equation 9 for kI in Equation 8 under paragraph (d)(1)(v) of this section, and rearranging, one obtains �(d[C]/[C]=kIo[exp(훛t)]dt+kDdt. This expression is integrated to give Equation 10: Equation 10 Pn(Co/C)SHW=(kIo/k)[1훒xp(훛t)]+kDt. The term (kIo/k) can now be evaluated. Since in pure water, Pn(Co/C)W=kDt, then subtracting this equation from Equation 10 gives Equation 11 Pn(Co/C)SHW-Pn(co/C)W=(kIo/k)[1-exp(-kt)]. The photobleaching fraction, [1-exp(-kt)], is equivalent to the expression [1-(A370/A�370)], where A�370 and A370 are the absorbances at 370 nm, and are proportional to humic sensitizer content at times zero and t. Therefore, (kIo/k) is derived from the slope of a linear regression using [Pn(Co/C)SHW-Pn(Co/C)W] as the dependent variable and [1-(A370/A�370)SHW] as the independent variable. (vii) To evaluate kIo, the parameter k has to be evaluated under standard sunlight conditions. Therefore, the photolysis rate constant for the PNAP/PYR actinometer (kA) is used to evaluate k by linear regression on Equation 12: Equation 12 Pn(A�370/A370)=(k/kA)Pn(Co/C)PNAP, where the slope is (k/kA) and the value of kA is calculated from the concentration of pyridine and the absorption of light by PNAP: kA=2.2(0.0169)[PYR]ka. Values of ka are listed in the following Table 1. Table 1--Day Averaged Rate Constant (ka)\1\ For Sunlight Absorption by PNAP as a Function of Season and Decadic Latitude\2\ 컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴 Season Latitude 컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴 Spring Summer Fall Winter 컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴 20N........................................... 515 551 409 327 30N........................................... 483 551 333 232 40N........................................... 431 532 245 139 50N........................................... 362 496 154 64 컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴 \1\ka=@ egaLg in the units of day -\1\, (Mill et al. (1982) under paragraph (f)(10) of this section). \2\For use in Equation 15 under paragraph (d)(2)(i) of this section. The value of kIo is then given by Equation 13: Equation 13 kIo=(kIo/k)(k/kA)kA. (viii) To obtain kD, determine the ratio (kD/kA) from a linear regression of Pn(Co/C)W versus Pn(Co/C)PNAP according to Equation 13a: Equation 13a Pn(Co/C)W=(kD/kA)Pn(Co/C)PNAP. The slope is (kD/kA), and kD is obtained by multiplication of this slope with the known value of kA: i.e., kD=(kD/kA)kA. (ix) Then, (kp)SHW values in SHW are determined by summing kD and KIo as follows: Equation 14 (kp)SHW=kIo+kD. (x) Finally, kpE is calculated from the precise relationship, Equation 5a: Equation 5a kpE=0.455(kp)SHW. (2) Procedure. (i) Using the test chemical photoreaction rate constant in round tubes, (kp) SHW, determined in Phase 2 under paragraph (c) of this section, and the absorption rate constant, k&agr;&&agr;&psgr;&psgr;&phgr;&ngr;1� found in Table 1, under paragraph (d)(1)(vii) of this section, calculate the molar pyridine concentration required by the PNAP/PYR actinometer using Equation 15: Equation 15 [PYR]/M=26.9[(kp) SHW/ka]. This pyridine concentration makes the actinometer rate constant match the test chemical rate constant. (A) The variable ka (= @ e ga Lg) is equal to the day-averaged rate constant for sunlight absorption by PNAP (USEPA (1984) under paragraph (f)(14) of this section; Mill et al. (1982) under paragraph (f)(10) of this section, Zepp and Cline (1977) under paragraph (f)(19) of this section) which changes with season and latitude. (B) The variable ka is selected from Table 1 under paragraph (d)(1)(vii) of this section for the season nearest the mid-experiment date of Phase 2 studies and the decadic latitude nearest the experimental site. (ii) Once [PYR] is determined, an actinometer solution is prepared by adding 1.00 mL of 1.0 x 10−2 M (0.165 gms/100 mL) PNAP stock solution (in CH3 CN solvent) and the required volume, V, of PYR to a 1 liter volumetric flask. The flask is then filled with distilled water to give 1 liter of solution. The volume V can be calculated from Equation 16: Equation 16 V/mL=[PYR]/0.0124. The PNAP/PYR solutions should be wrapped with aluminum foil and kept out of bright light after preparation. (iii) The following solutions should be prepared and individually added in 6.00 mL aliquots to 12/100 mm quartz sample tubes; 8 tubes should be filled with each solution: (A) PNAP/PYR actinometer solution. (B) Test chemical in pH 7.0, 0.010 M phosphate buffer. (C) Test chemcial in pH 7.0, 0.010 M phosphate buffer/SHW. (D) pH 7.0, 0.010 M phosphate buffer/SHW. Four tubes of each set are wrapped in foil and used as controls. (iv) The tubes are placed in the photolysis rack (Phase 2, Procedure) at 0900 hours on day zero, with the controls taped to the bottom of the rack. One tube of each composition is removed, along with their respective controls, according to a schedule found in Table 2, which categorizes sampling times on the basis of (kp)SHW determined in Phase 1. Table 2--Category and Sampling Procedure for Test and Actinometry Solutions 컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴 kp Category (d–1)SHW Sampling procedure 컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴 A.................................... 5.5 J Kp J 0.69 Sample at 0, 1, 2, 4, and 8h. B.................................... 0.69>kp J 0.017 Sample at 0, 1, 2, 4, and 8d. C.................................... 0.17>kp J 0.043 Sample at 0, 4, 8, 16, and 32d. 컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴 (v) The tubes containing PNAP, test chemical, and their controls are analyzed for residual concentrations soon after the end of the experiment. PNAP is conveniently analyzed by HPLC, using a 30 cm C18 reverse phase column and a uv detector set at 280 nm. The mobile phase is 2 percent acetic acid, 50 percent acetonitrile and 48 percent water (2 mL/min flow rate). Tubes containing only SHW (solution D) should be analyzed by absorption spectroscopy at 370 nm after storage at 4�C in the dark. The absorbance range to be measured is 0.05 to 0.01 AU (1 cm). (vi) If controls are well-behaved and show no significant loss of chemical or absorbance change, then kI can be calculated. In tabular form (see Table 4 under paragraph (d)(6)(iii)(A) of this section) arrange the quantities Pn(Co/Ct) SHW, Pn(Co/Ct)SHW, [1�(A370/Ao370)], Pn(Ao370/A370), and Pn(Co/C)PNAP in order of increasing time. According to Equation 11 under paragraph (d)(1)(vi) of this section in the form of Equation 17, Equation 17 Pn(Co/C)SHW횾n(Co/C)W=(kIo/k)[1�(A370/Ao370)], plot the quantities [Pn(Co/Ct)SHW횾n(Co/Ct)W] versus the independent variable [1�(A370/Ao370)]. Obtain the slope (S1) by least square linear regression. Under the assumptions of the protocol, S1=(kIo/k). (vii) According to Equation 12 under paragraph (d)(1)(vii) of this section, plot the quantities Pn(Ao370/A370) versus the independent variable Pn(Co/Ct)PNAP. Obtain the slope (S2) by least squares linear regression on Equation 12 under paragraph (d)(1)(vii) of this section. Under the assumptions of the protocol, S2=(k/kA). (viii) Then, using Equation 13a under paragraph (d)(1)(vii) of this section, determine the slope (S3) by least squares linear regression. Under the assumptions of the protocol, S3 is equal to (kD/kA). (ix) From Equation 18 Equation 18 kA=0.0372[PYR]ka, calculate kA using ka values found in Table 1 under paragraph (d)(1)(vii) of this section. The value of ka chosen must correspond to the date closest to the mid-experiment date and latitude closest to that of the experimental site. (x) The indirect photoreaction rate constant, kIo, is determined using Equation 19, Equation 19 kIo=(S1)(kA)(S2), by incorporating the quantities kA, S1, and S2 determined as described in paragraphs (d)(2) (ix), (vi), and (vii) of this section, respectively. (xi) The rate constant kD is calculated from Equation 20, Equation 20 kD=(S3)(kA), using the quantities S3 and kA determined as described above. (xii) Then, (kp)SHW is obtained by summing kD and kIo, as described by Equation 14 in paragraph (d)(1)(ix) of this section: Equation 14 (kp)SHW=kIo+kD. (xiii) Finally, kpE is obtained by multiplying (kp) SNW by the factor 0.455, as described by Equation 5a in paragraph (d)(1)(x) of this section: Equation 5a kpE=0.455 (kp) SHW As determined, kpE is the net environmental photoreaction rate constant. It applies to clear sky conditions and is valid for predicting surface photoreaction rates in an average humic containing freshwater body. It is strictly valid only for the experimental latitude and season. (3) Criteria for Phase 3. As in Phase 2, Phase 3 tests are assumed valid if the dark controls are well behaved and show no significant loss of chemical. In such a case, loss of test chemical in irradiated samples is due to photoreaction. (4) Rationale. Simultaneous irradiation of a test chemical and actinometer provide a means of evaluating sunlight intensities during the reaction period. Parallel irradiation of SHW solutions allows evaluation of the extent of photobleaching and loss of sensitizing ability of the natural water. (5) Scope and limitations of Phase 3 protocol. Test chemicals that are classified as having half-lives in SHW in the range of 1 hour to 50 days in Phase 2 listing are suitable for use in Phase 3 testing. Such chemicals have photoreaction half-lives in a range accommodated by the PNAP/PYR actinometry in sunlight and also accommodate the persistence of SHW in sunlight. (6) Illustrative example. (i) From Phase 2 testing, under paragraph (c)(6)(iii) of this section, chemical A was found to have a photolysis rate constant, (kp) SHW' of 0.30 d–1 in fall in round tubes at latitude 33+N. Using Table 1 under paragraph (d)(1)(vii) of this section for 30+N, the nearest decadic latitude, a fall value of ka equal to 333 d–1 is found for PNAP. Substitution of (kp)SHW and ka into Equation 15 under paragraph (d)(2)(i) of this section gives [PYR] = 0.0242 M. This is the concentration of pyridine that gives an actinometer rate constant of 0.30 d–1 in round tubes in fall at this latitude. (ii) The actinometer solution was made up by adding a volume of pyridine (1.95 mL) calculated from equation 16 under paragraph (d)(2)(ii) of this section to a 1 liter volumetric flask containing 1.00 mL of 1.00 x 10−2 M PNAP in acetonitrile. The flask was filled to the mark with distilled water to give final concentrations of [PYR]=0.0242 M and [PNAP]=1.00 x 10−5 M. Ten tubes of each of the following solutions were placed in the photolysis rack at 1,200 hours on day zero: (A) Chemical A (1.53 x 10−5M) in standard SHW (0.010 M, pH 7 phosphate buffer). (B) Chemical A (1.53 x 10−5), in 0.010 M, pH 7 phosphate buffer. (C) SHW standard solution diluted with water 0.90 to 1.00 to match solution A. (D) PNAP/PYR actinometer solution. Ten additional foil-wrapped controls of each mixture were taped to the bottom of the rack. (iii) The test chemical had been placed in category B, Table 2 under the paragraph (d)(2)(iv) of this section, on the basis of its Phase 2 rate constant under paragraph (c) of this section. Accordingly, two tubes of each irradiated solution and two tubes of each blank solution were removed at 0, 1, 2, 4, and 8 days at 1,200 hours. The averaged analytical results obtained at the end of the experiment are shown in the following Table 3. Table 3--Chemical Analytical Results for Illustrative Example, Phase 3 컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴 10\5\[C]SHW, 10\5\[C]W, 105 Day M M ASHW370 [PNAP], M 컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴 0............................ 1.53 1.53 0.0500 1.00 1............................ 1.03 1.40 0.0470 0.810 2............................ 0.760 1.30 0.0440 0.690 4............................ 0.300 1.01 0.0370 0.380 8............................ 0.130 0.800 0.0320 0.220 컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴 Data for solutions A through D are given in column 2 through 5, respectively. No significant chemical loss was found in the dark controls. (A) From these items the functions Pn(Co/C) SNW' Pn(Co/C)W' [1-- (A370/Ao370)SNW], Pn(Ao370/A370), and Pn(Co/C)PNAP were calculated, as shown in the following Table 4 which was derived from Table 3 under paragraph (d)(6)(iii) of this section: Table 4--Photoreaction Function for Illustrative Examples, Phase 3, Derived From Table 3 컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴 Pn(Ao Pn(Co Day Pn(Co/ Pn(Co/ 1-(A 370 / 370 / /C) C)SHW C)W Ao370) A370) PNAP 컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴 0......................................... 0 0 0 0 0 1............................................. 0.396 0.0888 0.0600 0.0618 0.211 2............................................. 0.700 0.163 0.120 0.128 0.371 4............................................. 1.629 0.415 0.260 0.301 0.968 8............................................. 2.465 0.648 0.360 0.446 1.514 컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴 (B) Slope S1=(kIo/k) was calculated according to Equation 17 under paragraph (d)(2)(vi) of this section and was found to be 4.96 by a least squares regression with a correlation coefficient equal to 0.9980. The following Figure 1 shows a plot of Equation 17 under paragraph (d)(2)(vi) of this section and its best-fit line. Insert Illus. 590A Figure 1--Graphic determination of S1=(kIo/k) based on Equation 17 under paragraph (d)(2)(vi) of this section. (C) Slope S2=(k/ka) was also derived from Table 4 under paragraph (d)(6)(iii)(A) of this section by a fit of Pn(Ao370 /A370) SHW and Pn(Co /C)PNAP to Equation 12 under paragraph (d)(l)(vii) of this section. This plot is displayed in the following Figure 2; the slope S2 was found to be 0.295 and the correlation coefficient was equal to 0.9986. Insert Illus. 592A Figure 2--Graphic determination of S2=(k/kA) based on Equation 12 under paragraph (d)(1)(vii) of this section. (D) Using the data in columns 3 and 6 in Table 4 under paragraph (d)(6)(iii)(A) of this section, slope S3 was calculated by regression from Equation 13a under paragraph (d)(1)(viii) of this section and was found to be 0.428 with correlation coefficient euqal to 0.99997. (E) Using Equation 18 under paragraph (d)(2)(ix) of this section, kA was found to be =0.300d−1. (F) The values of S1, S2, and kA were then combined in Equation 19 under paragraph (d)(2)(x) of this section to give kIo as follows: Equation 19 kIo=(4.96)(0.300)(0.295)=0.439d−1. (G) The rate constant kD was calculated from the product of S3 and kA as expressed in Equation 20 under paragraph (d)(2)(xi) of this section as follows: Equation 20 kD=(0.428)(0.300)=0.128d−1. (H) The sum of kD and kIo was multiplied by 0.455 to obtain kpE as follows: Equation 21 kpE=(0.455)(0.439+0.128)d−1=0.258d−1 . (I) Since kpE is a first-order rate constant, the half-life, t1/2E, is given by Equation 22: Equation 22 t1/2E=0.693/kpE. Substituting the value of kpE from Equation 21 under paragraph (d)(6)(iii)(H) of this section in Equation 22 yielded Equation 23 t1/2E=0.693/0.258d−1=2.7d. (e) Data and reporting--(1) Test conditions--(i) Specific analytical and recovery procedures. (A) Provide a detailed description or reference for the analytical procedures used, including the calibration data and precision. (B) If extraction methods were used to separate the solute from the aqueous solution, provide a description of the extraction method as well as the recovery data. (ii) Other test conditions. (A) Report the site and latitude where the photolysis experiments were carried out. (B) Report the dates of photolysis, weather conditions, times of exposure, and the duration of exposure. (C) If acetonitrile was used to solubilize the test chemical, report the volume percent. (D) If a significant loss of test chemical occurred in the control solutions for pure water and SHW, indicate the causes and how they were eliminated or minimized. (2) Test data report--(i) Phase 2 Screening Test under paragraph (c) of this section. (A) Report the initial molar concentration of test chemical, Co, in pure water and SHW for each replicate and the mean value. (B) Report the molar concentration of test chemical, Ct, in pure water and SHW for each replicate and the mean value for each time pointt. (C) Report the molar concentration of test chemical for each replicate control sample and the mean value for each time point. (D) Report the values of (kp)SHW and (kp)W for the time pointt in which the fraction of test chemical photoreacted is in the range 20 to 80 percent. (E) If small losses of test chemical were observed in SHW and pure water, report a first-order rate constant loss, (kp)loss. Calculate and report (kp)obs for SHW and/or pure water. Calculate and report the corrected first-order rate constant for SHW and/or pure water using the relationship expressed in Equation 24: Equation 24 kp=(kp)obs�(kp)l oss. (F) Report the value of R calculated from Equation 4 under paragraph (c)(2)(vi)(D)(4) of this section. (G) Report the values of kpE and kDE obtained from Equations 5 and 6, respectively under paragraph (c)(2)(vii) of this section; report the corresponding half-life calculated from Equation 22 under paragraph (d)(6)(iii)(I) of this section. (ii) Phase 3--Indirect photoreaction with actinometer. (A) Report the initial molar concentration of test chemical, Co, in pure water and in SHW for each replicate and the mean value. (B) Report the initial absorbance Ao370 of the SNW solution. (C) Report the initial molar concentration of PNAP of each replicate and the mean value in the actinometer. Report the concentration of pyridine used in the actinometer which was obtained from Equation 15 under paragraph (d)(2)(i) of this section. (D) Report the time and date the photolysis experiments were started, the time and date the experiments were completed, and the elapsed photolysis time in days. (E) For each time point t, report the separate values of the absorbance of the SHW solution, and the mean values. (F) For each time point for the controls, report the separate values of the molar concentrations of test chemical in pure water and SHW, and the absorbance of the SHW solution, and the mean values. (G) Tabulate and report the following data: t, [C]SHW, [C]W, ASNW370, [PNAP]. (H) From the data in (G), tabulate and report the following data: t, Pn(Co/C)SNW, Pn(Co/C)W, [1�(A370/Ao370)SNW], Pn(Ao370/A370), Pn(Co/C)PNAP. (I) From the linear regression analysis of the appropriate data in step (H) in Equation 17 under paragraph (d)(2)(vi) of this section, report the slope S1 and the correlation coefficient. (J) From the linear regression analysis of the appropriate data in step (H) in Equation 12 under paragraph (d)(1)(vii) of this section, report the slope S2 and the correlation coefficient. (K) From the linear regression analysis of the appropriate data in step (H) in Equation 13a under paragraph (d)(1)(viii) of this section, report the slope S3 and the correlation coefficient. (L) If loss of chemical was observed during photolysis in pure water and SHW, then report the data Pn(Co/C)corr, Pn(Co/C)obs, Pn(Co/C)loss as described in paragraph (e)(2)(E) of this section. Repeat steps (H), (I), (J), (K) where applicable and report S1, S2, S3 and the corresponding correlation coefficients. (M) Report the value of the actinometer rate constant obtained from Equation 18 under paragraph (d)(2)(ix) of this section. (N) Report the value of kIo obtained from Equation 19 under paragraph (d)(2)(x) of this section. (O) Report the value of kD obtained from Equation 20 under paragraph (d)(2)(xi) of this section. (P) Report the value of (kpE)SHW, obtained from Equation 14 under paragraph (d)(1)(ix) of this section, and the value of kpE obtained from Equation 5a under paragraph (d)(1)(x) of this section. (Q) Report the half-life, t1/2E, obtained from Equation 22 under paragraph (d)(6)(iii)(I) of this section. (f) References. For additional background information on this test guideline the following references should be consulted. (1) Cooper W.J., Zika R.G. ``Photochemical formation of hydrogen peroxide in surface and ground waters exposed to sunlight.'' Science, 220:711. (1983). (2) Draper W.M., Crosby D.G. ``The photochemical generation of hydrogen peroxide in natural waters.'' Archives of Environmental Contamination and Toxicology, 12:121. (1983). (3) Draper, W.M. and Crosby D.G. ``Solar photooxidation of pesticides in dilute hydrogen peroxide.'' Journal of Agricultural and Food Chemistry, 32:231. (1984). (4) Draper W.M., Crosby D.G. ``Hydrogen peroxide and hydroxyl radical: Intermediates in indirect photolysis reactions in water.'' Journal of Agricultural and Food Chemistry, 29:699. (1981). (5) Dulin D., Mill T. ``Development and evaluation of sunlight actinometers.'' Environmental Science and Technology, 6:815. (1982). (6) Haag H.R., Hoigne J., Gassman E., Braun A.M. ``Singlet oxygen in surface waters--Part I; Furfuryl alcohol as a trapping agent.'' Chemosphere, 13:631. (1984). (7) Haag W.R., Hoigne J., Gassman E., Braun A.M. ``Singlet oxygen in surface waters--Part II: Quantum yields of its production by some natural humic materials as a function of wavelength.'' Chemosphere, 13:641. (1984). (8) Mill T., Winterle J.S., Fischer A., Tse D., Mabey W.R., Drossman H., Liu A., Davenport J.E. Toxic substances process data generation and protocol development. Work assignment 12, test standard development. ``Section 3. Indirect photolysis.'' Draft final report. EPA Contract No. 68 - 03 - 2981. Environmental Research Laboratory, Office of Research and Development, EPA, Athens, GA, and Office of Pollution Prevention and Toxics, EPA, Washington, DC. (1984). (9) Mill T., Mabey W.R., Bomberger D.C., Chou T.W., Hendry D.G., Smith J.H. ``Laboratory protocols for evaluating the fate of organic chemicals in air and water. Chapter 3. Photolysis in water. Chapter 4. Oxidation in water.'' EPA 600/3 - 82 - 022. Environmental Research Laboratory, Office of Research and Development, EPA, Athens, GA. (1981). (10) Mill T., Mabey W.R., Winterle J.S., Davenport J.E., Barich V.P., Dulin D.E., Tse D.S., Lee G. ``Design and validation of screening and detailed methods for environmental processes. Apendix C. Lower-tier direct photolysis protocol.'' Draft final report. EPA Contract No. 68 - 01 - 6325. Office of Pollution Prevention and Toxics, EPA, Washington, DC. (1982). (11) Mill T., Davenport J.E., Winterle J.S., Mabey W.R., Dossman H., Tse D., Liu A. Toxic substances process data generation and protocol development. Work assignment 12. ``Appendix B. Upper-tier protocol for direct photolysis in water.'' Draft final report. EPA Contract No. 68 - 03 - 2981. Environmental Research Laboratory, Office of Research and Development, EPA, Athens, GA, and Office of Pollution Prevention and Toxics, EPA, Washington, DC. (July 1983). (12) Winterle J.S., Mill T. Toxic substances process data generation and protocol development. Work assignment 18. ``Indirect photoreaction protocol.'' Draft EPA special report. EPA Contract No. 68 - 03 - 2981. Environmental Research Laboratory, Office of Research and Development, EPA, Athens, GA and Office of Pollution Prevention and Toxics, EPA, Washington, DC. (1985). (13) Mill T., Hendry D.G., Richardson H. ``Free radical oxidants in natural waters.'' Science, 207:886. (1980). (14) U.S. Environmental Protection Agency (USEPA), Office of Pollution Prevention and Toxics (OPPT). ``Chemical fate test guidelines. Test guideline (CG, CS - 6000). Photolysis in aqueous solution.'' EPA - 560/6 - 84 - 003. NTIS publication PB - 84 - 233287. (1984). (15) USEPA, OPPT. ``Chemical fate test guidelines. Test guildeline (CG, CS - 6010). Laboratory determination of the direct photolysis reaction quantum yield in aqueous solution and sunlight photolysis.'' EPA - 560/6 - 84 - 003. NTIS publication PB - 84 - 233287. (1984). (16) Wolff C.J.M., Halmans M.T.H., Van der Heijde H.B. ``The formation of singlet oxygen in surface waters.'' Chemosphere, 10:59. (1981). (17) Zepp R.G., Baughman G.L., Schlotzhauer P.F. ``Comparison of photochemical behavior of various humic substances in water: I. Sunlight induced reactions of aquatic pollutants photosensitized by humic substances.'' Chemosphere, 10:109. (1981). (18) Zepp R.G., Baughman G.L., Schlozhauer P.F. ``Comparison of photochemical behavior of various humic substances in water: II. Photosensitized oxygenations.'' Chemosphere, 10:119. (1981). (19) Zepp R.G., Cline D.M. ``Rates of direct photolysis in aquatic environments.'' Environmental Science and Technology, 11:359. (1977). (20) Zepp, R.G., Wolfe N.L., Baughman G.L., Hollis R.C. ``Singlet oxygen in natural waters.'' Nature, 267:421. (1977). (21) Zepp R.G., Schlotzhauer P.F., Merritt S.R. ``Photosensitized transformations involving electronic energy transfer in natural waters: role of humic substances.'' Environmental Science and Technology, 19:74. (1985). [53 FR 34522, Sept. 7, 1988; 53 FR 37393, Sept. 26, 1988] Subpart C--Provisional Environmental Effects Guidelines 795.120 Gammarid acute toxicity test. (a) Purpose. This guideline is intended for use in developing data on the acute toxicity of chemical substances and mixtures subject to environmental effects test regulations under the Toxic Substances Control Act (TSCA) (Pub. L. 94 - 469, 90 Stat. 2003 (15 U.S.C. 2601 et. seq.)). This guideline describes a test to develop data on the acute toxicity of chemicals to gammarids. The United States Environmental Protection Agency (EPA) will use data from this test in assessing the hazard of a chemical to aquatic organisms. (b) Definitions. The definitions in section 3 of TSCA and in part 792 of this chapter, Good Laboratory Practice Standards, apply to this test guideline. The following definitions also apply to this guideline: ``Death'' means the lack of reaction of a test organism to gentle prodding. ``Flow-through'' means a continuous or an intermittent passage of test solution or dilution water through a test chamber or a holding or acclimation tank, with no recycling. ``LC50'' means the median lethal concentration, i.e., that concentration of a chemical in air or water killing 50 percent of the test batch of organisms within a particular period of exposure (which shall be stated). ``Loading'' means the ratio of the biomass of gammarids (grams, wet weight) to the volume (liters) of test solution in either a test chamber or passing through it in a 24-hour period. ``Solvent'' means a substance (e.g., acetone) which is combined with the test substance to facilitate introduction of the test substance into the dilution water. ``Static system'' means a test chamber in which the test solution is not renewed during the period of the test. (c) Test procedures--(1) Summary of the test. In preparation for the test, test chambers are filled with appropriate volumes of dilution water. If a flow-through test is performed, the flow of dilution water through each chamber is adjusted to the rate desired. In a static test, the test substance is introduced into each test chamber. In a flow-through test, the rate in which the test substance is added is adjusted to establish and maintain the desired concentration of test substance in each test chamber. The test is started by randomly introducing gammarids, which have been acclimated to the test conditions, into the test chambers. Gammarids in the test chambers are observed periodically during the test; the dead gammarids are removed and the findings recorded. Dissolved oxygen concentration, pH, temperature, and the concentration of test substance in test chambers are measured at specified intervals. Data collected during the test are used to develop concentration--response curves and LC50 values for the test substance. (2) [Reserved] (3) Range-finding test. (i) A range-finding test should be conducted to establish test substance concentrations to be used for the definitive test. (ii) The gammarids shall be exposed to a wide-range of concentrations of the test substance (e.g., 1, 10, 100 mg/1, etc.), usually under static conditions. (iii) A minimum of five gammarids should be exposed to each concentration of test substance for a period of 96 hours. The exposure period may be shortened if data suitable for determining concentrations in the definitive test can be obtained in less time. Nominal concentrations of the test substance may be acceptable. (4) Definitive test. (i) The purpose of the definitive test is to determine the 24, 48, 72, and 96--hour LC50 values and the concentration-response curves. (ii) A minimum of 20 gammarids per concentration shall be exposed to five or more concentrations of the test substance chosen in a geometric series in which the ratio is between 1.5 and 2.0 (e.g., 2, 4, 8, 16, 32, 64 mg/L). The range and number of concentrations to which the organisms are exposed shall be such that in 96 hours there is at least one concentration resulting in mortality greater than 50 and less than 100 percent, and one concentration causing greater than zero and less than 50 percent mortality. An equal number of gammarids may be placed in two or more replicate test chambers. Solvents should be avoided, if possible. If solvents have to be used, a solvent control, as well as a dilution control, shall be tested at the highest solvent concentration employed in the treatments. The solvent should not be toxic or have an effect on the toxicity of the test substance. The concentration of solvent should not exceed 0.1 ml/L. (iii) Every test shall include a concurrent control using gammarids from the same population or culture container. The control group shall be exposed to the same dilution water, conditions and procedures, except that none of the test substance shall be is added to the chamber. (iv) The dissolved oxygen concentration, temperature and pH of the test solution shall be measured at the beginning of the test and at 24, 48, 72 and 96 hours in at least one replicate each of the control, and the highest, lowest and middle test concentrations. (v) The test duration is 96 hours. The test is unacceptable if more than 10 percent of the control organisms die during the test. (vi) In addition to death, any abnormal behavior or appearance shall also be reported. (vii) Gammarids shall be randomly assigned to the test chambers. Test chambers shall be positioned within the testing area in a random manner or in a way in which appropriate statistical analyses can be used to determine whether there is any variation due to placement. (viii) Gammarids shall be introduced into the test chambers after the test substance has been added. (ix) Observations on compound solubility shall be recorded. The investigator should record the appearance of surface slicks, precipitates, or material adhering to the sides of the test chambers. (5) [Reserved] (6) Analytical measurements--(i) Water quality analysis. The hardness, acidity, alkalinity, pH, conductivity, TOC or COD, and particulate matter of the dilution water shall be measured at the beginning of each definitive test. (ii) Collection of samples for measurement of test substance. Each sample to be analyzed for the test substance concentrations shall be taken at a location midway between the top, bottom, and sides of the test chamber. Samples should not include any surface scum or material dislodged from the bottom or sides. Samples shall be analyzed immediately or handled and stored in a manner which minimizes loss of test substance through microbial degradation, photogradation, chemical reaction, volatilization, or sorption. (iii) Measurement of test substance. (A) For static tests, the concentration of dissolved test substance (that which passes through a 0.45 micron filter) shall be measured in each test chamber at least at the beginning (zero-hour, before gammarids are added) and at the end of the test. During flow-through tests, the concentration of dissolved test substance shall be measured in each test chamber at least at 0 and 96-hours and in at least one chamber whenever a malfunction of the test substance delivery system is observed. (B) The analytical methods used to measure the amount of test substance in a sample shall be validated before beginning the test. This involves adding a known amount of the test substance to each of three water samples taken from a chamber containing dilution water and the same number of gammarids as are placed in each test chamber. The nominal concentrations of the test substance in these samples should span the concentration range to be used in the test. Validation of the analytical method should be performed on at least two separate days prior to starting the test. (C) An analytical method is not acceptable if likely degradation products of the test substance give positive or negative interferences, unless it is shown that such degradation products are not present in the test chambers during the test. (D) Among replicate test chambers, the measured concentrations shall not vary more than 20 percent. The measured concentration of the test substance in any chamber during the test shall not vary more than plus or minus 30 percent from the measured concentration in that chamber at zero time. (E) The mean measured concentration of dissolved test substance shall be used to calculate all LC50's and to plot all concentration-response curves. (d) Test conditions for definitive test--(1) Test species--(i) Selection. (A) The amphipods, Gammarus fasciatus, G. pseudolimnaeus, and G. lacustris are specified for this test. (B) Gammarids can be cultured in the laboratory or collected from natural sources. If collected, they must be held in the laboratory for at least 14 days prior to testing. (C) Gammarids used in a particular test shall be of similar age and/or size and from the same source or culture population. (ii) Acclimation. If the holding water is from the same source as the dilution water, acclimation to the dilution water shall be done gradually over a 48-hour period. The gammarids then shall be held at least 7 days in the dilution water prior to testing. Any changes in water temperature should not exceed 2 +C per day. Gammarids should be held for a minimum of 7 days at the test temperature prior to testing. (iii) Care and handling. Gammarids shall be cultured in dilution water under similar environmental conditions to those used in the test. Organisms shall be handled as little as possible. When handling is necessary it should be done as gently, carefully and quickly as possible. During culturing and acclimation, gammarids shall be observed carefully for signs of stress and mortality. Dead and abnormal individuals shall be discarded. (iv) Feeding. The organisms shall not be fed during testing. During culturing, holding, and acclimation, a sufficient quantity of deciduous leaves, such as maple, aspen, or birch, should be placed in the culture and holding containers to cover the bottom with several layers. These leaves should be aged for at least 30 days in a flow-through system before putting them in aquaria. As these leaves are eaten, more aged leaves should be added. Pelleted fish food may also be added. (2) Facilities--(i) Apparatus--(A) Facilities needed to perform this test include: (1) Containers for culturing, acclimating and testing gammarids; (2) Containers for aging leaves under flow-through conditions; (3) A mechanism for controlling and maintaining the water temperature during the culturing, acclimation and test periods; (4) Apparatus for straining particulate matter, removing gas bubbles, or aerating the dilution water, as necessary; and (5) An apparatus for providing a 16-hour light and 8-hour dark photoperiod with a 15- to 30-minute transition period. (B) Facilities should be well ventilated and free of fumes and disturbances that may affect the test organism. (C) Test chambers shall be covered loosely to reduce the loss of test solution or dilution water due to evaporation and to minimize the entry of dust or other particulates into the solutions. (ii) Construction materials. Construction materials and equipment that may contact the stock solution, test solution or dilution water should not contain substances that can be leached or dissolved into aqueous solutions in quantities that can alter the test results. Materials and equipment that contact stock or test solutions should be chosen to minimize sorption of test substances. Glass, stainless steel, and perfluorocarbon plastic should be used wherever possible. Concrete, fiberglass, or plastic (e.g., PVC) may be used for holding tanks, acclimation tanks, and water supply systems, but they should be aged prior to use. Rubber, coopper, brass, galvanized metal, and lead should not come in contact with the dilution water, stock solution, or test solution. (iii) Test substance delivery system. In flow-through tests, diluters, metering pump systems or other suitable devices shall be used to deliver the test substance to the test chambers. The system used shall be calibrated before each test. The general operation of the test substance delivery system shall be checked twice daily during a test. The 24-hour flow shall be equal to at least five times the volume of the test chamber. During a test, the flow rates should not vary more than 10 percent from one test chamber to another. (iv) Test chambers. Test chambers shall contain at least one liter of test solution. Test chambers made of stainless steel should be welded, not soldered. Test chambers made of glass should be glued using clear silicone adhesive. As little adhesive as possible should be left exposed in the interior of the chamber. A substrate, such as a bent piece of stainless steel screen, should be placed on the bottom of each test chamber to provide cover for the gammarids. (v) Cleaning of test system. Test substance delivery systems and test chambers should be cleaned before each test. They should be washed with detergent and then rinsed sequentially with clean water, pesticide-free acetone, clean water, and 5-percent nitric acid, followed by two or more changes of dilution water. (vi) Dilution water. (A) Clean surface or ground water, reconstituted water, or dechlorinated tap water is acceptable as dilution water if gammarids will survive in it for the duration of the culturing, acclimating, and testing periods without showing signs of strees. The quality of the dilution water should be constant enough that the month-to-month variation in hardness, acidity, alkalinity, conductivity, TOC or COD, and particulate matter is not more than 10 percent. The pH should be constant within 0.4 unit. In addition, the dilution water should meet the following specifications measured at least twice a year: 컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴 Maximum Substance concentration 컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴 Particulate matter............................................. 20 mg/L Total organic carbon (TOC) or.................................. 2 mg/L chemical oxygen demand (COD)................................. 5 mg/L Boron, fluoride................................................ 100 ug/L Un-ionized ammonia............................................. 1 ug/L Aluminum, arsenic, chromium, cobalt, copper, iron, lead, nickel, zinc.................................................. 1 ug/L Residual chlorine.............................................. 3 ug/L Cadmium, mercury, silver....................................... 100 ng/L Total organophosphorus pesticides.............................. 50 ng/L Total organochlorine pesticides plus: polychlorinated biphenyls (PCBs) or.......................... 50 ng/L organic chlorine............................................. 25 ng/L 컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴컴 (B) If the dilution water is from a ground or surface water source, conductivity and total organic carbon (TOC) or chemical oxygen demand (COD) shall be measured. Reconstituted water can be made by adding specific amounts of reagent-grade chemicals to deionized or distilled water. Glass-distilled or carbon-filtered deionized water with a conductivity less than 1 micromho/cm is acceptable as the diluent for making reconstituted water. (C) The concentration of dissolved oxygen in the dilution water shall be between 90 and 100 percent saturation. If necessary, the dilution water can be aerated before the addition of the test substance. All reconstituted water should be aerated before use. (3) Test parameters. Environmental parameters during the test shall be maintained as specified below: (i) Water temperature of 18 ÷ 1�C. (ii) Dissolved oxygen concentration between 60 and 105 percent saturation. (iii) The number of gammarids placed in a test chamber shall not be so great as to affect the results of the test. Ten gammarids per liter is the recommended level of loading for the static test. Loading requirements for the flow-through test will vary depending on the flow rate of dilution water. The loading should not cause the dissolved oxygen concentration to fall below the recommended levels. (iv) Photoperiod of 16 hours light and 8 hours darkness. (e) Reporting. The sponsor shall submit to the EPA all data developed by the test that are suggestive or predictive of toxicity. In addition, the test report shall include, but not necessarily be limited to, the following information: (1) Name and address of the facility performing the study and the dates on which the study was initiated and completed. (2) Objectives and procedures stated in the approved protocol, including any changes in the original protocol. (3) Statistical methods employed for analyzing the data. (4) The test substance identified by name, Chemical Abstracts (CAS) number or code number, source, lot or batch number, strength, purity, and composition, or other appropriate characteristics. (5) Stability of the test substance under the conditions of the test. (6) A description of the methods used, including: (i) The source of the dilution water, its chemical characteristics (e.g., hardness, pH, etc.) and a description of any pretreatment. (ii) A description of the test substance delivery system, test chambers, the depth and volume of solution in the chamber, the way the test was begun (e.g., test substance addition), the loading, the lighting, and the flow rate. (iii) Frequency and methods of measurements and observations. (7) The scientific name, weight, length, source, and history of the organisms used, and the acclimation procedures and food used. (8) The concentrations tested, the number of gammarids and replicates per test concentration. The reported results should include: (i) The results of dissolved oxygen, pH and temperature measurements. (ii) If solvents are used, the name and source of the solvent, the nominal concentration of the test substance in the stock solution, the highest solvent concentration in the test solution and a description of the solubility determination in water and solvents. (iii) The measured concentration of the test substance in each test chamber just before the start of the test and at all subsequent sampling periods. (iv) In each test chamber at each observation period, the number of dead and live test organisms, the percentage of organisms that died, and the number of test organisms that showed any abnormal effects in each test chamber at each observation period. (v) The 48, 72 and 96-hour LC50's and their 95 percent confidence limits. When sufficient data have been generated, the 24-hour LC50 value also. These calculations should be made using the mean measured test substance concentrations. (vi) The observed no-effect concentration (the highest concentration tested at which there were no mortalities or abnormal behavioral or physiological effects), if any. (vii) Methods and data for all chemical analyses of water quality and test substance concentrations, including method validations and reagent blanks. (9) A description of all circumstances that may have affected the quality or integrity of the data. (10) The names of the sponsor, study director, principal investigator, names of other scientists or professionals, and the names of all supervisory personnel involved in the study. (11) A description of the transformations, calculations, or operations performed on the data, a summary and analysis of the data, and a statement of the conclusions drawn from the analysis. Results of the analysis of data should include the calculated LC50 value, 95 percent confidence limits, slope of the transformed concentration-response line, and the results of a goodness-of-fit test (e.g., chi-square test). (12) The signed and dated reports prepared by any individual scientist or other professional involved in the study, including each person who, at the request or direction of the testing facility or sponsor, conducted an analysis or evaluation of data or specimens from the study after data generation was completed. (13) The locations where all specimens, raw data, and the final report are stored. (14) The statement prepared and signed by the quality assurance unit. [52 FR 24462, July 1, 1987] Subpart D--Provisional Health Effects Guidelines 795.223 Pharmacokinetic test. (a) Purpose. The purpose of these tests is to determine: (1) The bioavailability of a test substance after dermal administration. (2) Whether or not the biotransformation of the test substance is qualitatively and quantitatively the same after dermal and oral administration. (3) Whether or not the biotransformation of the test substance is changed qualitatively or quantitatively by repeated dosing. (b) Definitions--(1) Bioavailability refers to the rate and extent to which the administered compound is absorbed, i.e., reaches the systemic circulation. (2) Relative percent of percutaneous absorption is defined as 100 times the ratio between total urinary excretion of compound following topical administration and total urinary excretion of compound following intravenous injection. (c) Test procedures--(1) Animal selection--(i) Species. The species utilized for investigating the test substance shall be the rat, a species for which historical data on the toxicity and carcinogenicity of several compounds are available and which is used extensively in percutaneous absorption studies. (ii) Animals. Adult female Fischer 344 rats shall be used. The rats shall be 7 to 9 weeks old and weigh 125 to 175 grams. Prior to testing the animals shall be selected at random for each group. Animals showing signs of ill health shall not be used. (iii) Animal care. (A) The animals should be housed in environmentally controlled rooms with 10 to 15 air changes per hour. The rooms should be maintained at a temperature of 25÷2� C and humidity of 50÷10 percent with a 12-hour light/dark cycle per day. The rats should be kept in a quarantine facility for at least 7 days prior to use. (B) During the acclimatization period, the rats should be housed in cages on hardwood chip bedding. All animals shall be provided with conventional laboratory diets and water ad libitum. (2) Administration of test substance--(i) Test compound. Test studies require the use of both nonradioactive test substance and 14C-labeled test substance. Both preparations are needed to investigate under paragraph (a)(2) of this section. The use 14C-test substance is required to investigate under paragraphs (a) (1), (2), and (3) of this section because it will facilitate the work, improve the reliability of quantitative determinations, and increase the probability of observing the presence of previously unidentified metabolities. (ii) Dosage and treatment. (A) Two doses shall be used in the study, a ``low'' dose and a ``high'' dose. When administered orally, the ``high'' dose level should ideally induce some overt toxicity such as weight loss. The ``low'' dose level should correspond to a no-effect level. (B) The same ``high'' and ``low'' doses shall be administered orally and dermally. (C) Oral dosing shall be performed by gavage or by administering encapsulated test substance. (D) For dermal treatment, the doses shall be applied at a volume adequate to deliver the prescribed doses. The backs of the rats should be lightly shaved with an electric clipper shortly before treatment. The dose shall be applied with a micropipette on 2 cm2 of the freshly shaven skin. The dosed areas shall be occluded with an aluminum foil patch which is secured in place with adhesive tape. (iii) Bioavailability study in rats. At least eight rats shall receive a single intravenous (low) dose of 14C-test substance and serial samples of blood removed from four animals at 15 minutes, 30 minutes, 1 hour, 8 hours, 24 hours, 48 hours, and 96 hours. All animals shall be housed in metabolism cages and urine and feces collected at 8, 24, 48, 72, and 96 hours. The procedure shall be repeated with eight rats in which 14C-test substance is maintained in contact with the skin for the duration of the study (96 hours). If dermal absorption cannot be demonstrated, the study should be repeated using a higher dose. Total radioactivity shall be measured in the blood, urine, and feces samples collected from all animals. The results shall be used to construct a blood concentration-time curve and to calculate bioavailability by the ratio of the total 96-hour urinary excretion of radioactivity after dermal and intravenous administration. Bioavailability is expressed as (percent dose dermal/percent dose intravenous) x 100=percent dermal absorption. Urine shall be saved for metabolite identification, if it becomes necessary. (iv) Biotransformation in rats after oral and dermal administration. Eight rats shall be dosed orally, and eight rats shall be dosed dermally (96-hour contact) with the high dose of 14C-test substance. The results of the bioavailability study (see paragraph (c)(2)(iii) of this section) shall be evaluated first to ensure that the dermal dose applied will result in the appearance of radioactivity in the urine. All animals shall be housed in metabolism cages allowing for separate collection of urine and feces at 8, 24, 48, 72, and 96 hours. The parent compound and any metabolite that comprises greater than 10 percent of the dose shall be identified in the urine. These results shall be qualitatively compared to the urinary excretion data obtained in the low dose bioavailability study (see paragraph (c)(2)(iii) of this section); metabolites in the low dose urine shall also be identified if a different pattern of metabolism is evident. (v) Repeated dosing study. Four rats shall receive a series of single daily oral doses of nonradioactive test substance over a period of at least 14 days, followed at 24 hours after the last dose by a single oral dose of 14C-test substance. Each dose shall be at the low-dose level. If the pattern of urinary metabolite excretion is qualitatively different from that obtained with the orally dosed animals in the single-dose biotransformation study at 24 and 48 hours (see paragraph (c)(2)(iv) of this section), metabolites shall be identified in accordance with the procedure given in paragraph (c)(2)(iii) of this section. (vi) Skin washing study. If greater than 10 percent of test substance is absorbed through the skin (see paragraphs (c)(2) (ii) and (iii) of this section) then a washing efficacy experiment shall be performed to assess the extent of removal of the applied test substance by washing with soap and water. Four rats should be lightly anesthetized and treated with a dermal dose of test compound previously shown to result in measurable percutaneous absorption greater than 10 percent. Soon after application (5 to 10 minutes) the treated animals shall be washed with soap and water, then housed in individual metabolism cages for excreta collection. Measurements of total radioactivity in urine and feces shall be made in the same manner as described in paragraph (c)(2)(iii) of this section. (d) Data and Reporting--(1) Treatment of results. Data shall be summarized in tabular form. (2) Evaluation of results. All observed results, quantitative or incidental, shall be evaluated by an appropriate statistical method. (3) Test report. In addition to the reporting requirements as specified in the TSCA Good Laboratory Practice Standards, 40 CFR part 792, subpart J, the following specific information shall be reported: (i) Species, strain, and supplier of laboratory animals. (ii) Information on the degree (i.e., specific activity for a radiolabel) and site(s) of labeling of the test substances. (iii) A full description of the sensitivity and precision of all procedures used to produce the data. (iv) Relative percent absorption by the dermal route for rats administered low and high doses of \14\C-test substance, compared with 100 percent of the intravenous dose. (v) Quantity of isotope, together with percent recovery of the administered dose, in feces, urine, and blood. (vi) Biotransformation pathways and quantities of the test substance and metabolites in urine collected after administering single high and low oral and dermal doses. (vii) Biotransformation pathways and quantities of test substance and metabolites in urine collected after administering repeated low doses of test substance to rats. [51 FR 40327, Nov. 6, 1986, as amended at 52 FR 24157, June 29, 1987] 795.225 Dermal pharmacokinetics of DGBE and DGBA. (a) Purpose. The purpose of these studies is to determine: (1) The absorption of diethylene glycol butyl ether (DGBE) after administration by the dermal route. (2) The biotransformation of DGBE administered dermally. (3) The dermal absorption of DGBE and diethylene glycol butyl ether acetate (DGBA). (b) Test procedures--(1) Animal selection--(i) Species. The species utilized for investigating DGBE and DGBA shall be the rat, a species for which historical data on the toxicity and carcinogenicity of many compounds are available and which is used extensively in percutaneous absorption studies. (ii) Animals. Adult female Sprague Dawley rats shall be used. The rats shall be 7 to 8 weeks old and weigh 180 to 220 grams. Prior to testing, the animals shall be selected at random for each group. Animals showing signs of ill health shall not be used. (iii) Animal care. (A) The animals should be housed in environmentally controlled rooms with 10 to 15 air changes per hour. The rooms should be maintained at a temperature of 25 ÷ 2�C and humidity of 50 ÷10 percent with a 12-hour light/dark cycle per day. The rats should be isolated for at least 7 days prior to use. (B) During the acclimatization period, the rats should be housed in cages on hardwood chip bedding. All animals shall be provided with conventional laboratory diets and water ad libitum. (2) Administration of DGBE and DGBA--(i) Test substances. These studies require the use of 14C-labeled DGBE and DGBA. The use of 14C-DGBE and 14C-DGBA is required for the determinations in paragraphs (a) (1), (2), and (3) of this section because they will facilitate the work and improve the reliability of quantitative determinations. (ii) Dosage and treatment. (A) Two doses of DGBA shall be used in the study, a ``low'' dose and a ``high'' dose. Three doses of DGBE shall be used in the study, a neat ``low'' dose, an aqueous ``low'' dose, and neat ``high'' dose. When administered dermally, the ``high'' dose level should ideally induce some overt toxicity such as weight loss. The ``low'' dose level should correspond to a no observed effect level. (B) For dermal treatment, the doses shall be applied in a volume adequate to deliver the prescribed doses. The backs of the rats should be lightly shaved with an electric clipper shortly before treatment. The dose shall be applied with a micropipette on a specific area (for example, 2 cm2) on the freshly shaven skin. (iii) Washing efficiency study. Before initiation of the dermal absorption studies described in paragraph (b)(2)(iv)(A) of this section, an initial washing efficiency experiment shall be performed to assess the extent of removal of the applied DGBE and DGBA by washing with soap and water. Groups of four rats should be lightly anesthetized with sodium pentobarbital. These animals shall then be treated with dermal doses of test substance at the low dose level. Soon after application (5 to 10 minutes) the treated animals shall be washed with soap and water then housed in individual metabolism cages for excreta collection. Urine and feces shall be collected at 8, 24, and 48 hours following dosing. Collection of excreta shall continue every 24 hours if a significant amounts of DGBE, DGBA, or metabolites continue to be eliminated. (iv) Determination of absorption, biotransformation, and excretion. (A) Eight animals shall be dosed once dermally with the low dose of 14C-DGBE. (B) Eight animals shall be dosed once dermally with the high dose of 14C-DGBE. (C) Eight animals shall be dosed once dermally with the low dose of 14C-DGBA. (D) Eight animals shall be dosed once dermally with the high dose of 14C-DGBA. (E) The high and low doses of 14C-DGBE and 14C-DGBA shall be kept on the skin for 24 hours. After application, the animals shall be placed in metabolism cages for excreta collection. After 24 hours, any test material remaining on the skin will be washed off and the containment cell removed. Radiolabeled material in the wash will be accounted for in the total recovery. Urine and feces shall be collected at 8, 24, 48, 72, and 96 hours after dosing, and if necessary, daily thereafter until at least 90 percent of the dose has been excreted or until 7 days after dosing, whichever occurs first. (3) Observation of animals--(i) Urinary and fecal excretion. The quantities of total 14C excreted in urine and feces by rats dosed as specified in paragraph (b)(2)(iv) of this section shall be determined at 8, 24, 48, 72 and 96 hours after dosing, and if necessary, daily thereafter until at least 90 percent of the dose has been excreted or until 7 days after dosing (whichever occurs first). Four animals from each group shall be used for this purpose. (ii) Biotransformation after dermal dosing. Appropriate qualitative and quantitative methods shall be used to assay urine specimens collected from rats dosed with DGBE as specified in paragraph (b)(2)(iv) of this section. Any metabolite which comprises greater than 10 percent of the dose shall be identified. (c) Data and reporting--(1) Treatment of results. Data shall be summarized in tabular form. (2) Evaluation of results. All observed results, quantitative or incidental, shall be evaluated by an appropriate statistical method. (3) Test report. In addition to the reporting requirements as specified in the TSCA Good Laboratory Practice Standards, in part 792, subpart J of this chapter, the following specific information shall be reported: (i) Species, strain, and supplier of laboratory animals. (ii) Information on the degree (i.e., specific activity for a radiolabel) and sites of labeling of the test substances. (iii) A full description of the sensitivity and precision of all procedures used to produce the data. (iv) Relative percent absorption by the dermal route for rats administered low and high doses of 14C-DGBE and 14C-DGBA. (v) Quantity of isotope, together with percent recovery of the administered dose, in feces and urine. (vi) Biotransformation pathways and quantities of DGBE and metabolites in urine collected after administering single high and low dermal doses to rats. [53 FR 5946, Feb. 26, 1988, as amended at 54 FR 41834, Oct. 12, 1989] 795.228 Oral/dermal pharmacokinetics. (a) Purpose. The purposes of these studies are to: (1) Ascertain whether the pharmacokinetics and metabolism of a chemical substance or mixture (``test substance'') are similar after oral and dermal administration. (2) Determine bioavailability of a test substance after oral and dermal administration. (3) Examine the effects of repeated dosing on the pharmacokinetics and metabolism of the test substance. (b) Definitions. (1) ``Bioavailability'' refers to the rate and relative amount of administered test substance which reaches the systemic circulation. (2) ``Metabolism'' means the study of the sum of the processes by which a particular substance is handled in the body and includes absorption, tissue distribution, biotransformation, and excretion. (3) ``Percent absorption'' means 100 times the ratio between total excretion of radioactivity following oral or dermal administration and total excretion following intravenous administration of test substance. (4) ``Pharmacokinetics'' means the study of the rates of absorption, tissue distribution, biotransformation, and excretion. (c) Test procedures--(1) Animal selection--(i) Species. The rat shall be used for pharmacokinetics testing because it has been used extensively for metabolic and toxicological studies. For dermal bioavailability studies, the rat and the mini-pig shall be used. (ii) Test animals. For pharmacokinetics testing and dermal studies, adult male and female Sprague - Dawley rats, 7 to 9 weeks of age, shall be used. For dermal studies, young adult mini-pigs shall also be used. The animals should be purchased from a reputable dealer and shall be identified upon arrival at the testing laboratory. The animals shall be selected at random for the test groups and any animal showing signs of ill health shall not be used. In all studies, unless otherwise specified, each test group shall contain at least 4 animals of each sex for a total of at least 8 animals. (iii) Animal care. (A) The animals shall be housed in environmentally controlled rooms with at least 10 air changes per hour. The rooms shall be maintained at a temperature of 24 ÷ 2 �C and humidity of 50 ÷ 20 percent with a 12-hour light/dark cycle per day. The animals shall be kept in a quarantine facility for at least 7 days prior to use and shall be acclimated to the experimental environment for a minimum of 48 hours prior to administration of the test substance. (B) During the acclimatization period, the animals shall be housed in suitable cages. All animals shall be provided with certified feed and tap water ad libitum. The mini-pig diet shall be supplemented with adequate amounts of ascorbic acid in the drinking water. (2) Administration of test substance--(i) Test substance. The use of a radioactive test substance is required for all studies. Ideally, the purity, radioactive and nonradioactive, is greater than 99 percent. The radioactive and nonradioactive test substances shall be chromatographed separately and together to establish purity and identity. If the purity is less than 99 percent or if the chromatograms differ significantly, EPA should be consulted. (ii) Dosage and treatment--(A) Intravenous. The low dose of test substance, in an appropriate vehicle, shall be administered intravenously to groups of rats and mini-pigs of each sex. If feasible, the same low dose should be used for intravenous, oral, and dermal studies. (B) Oral. Two doses of text substance shall be used in the oral study, a low dose and a high dose. The high dose should ideally induce some overt toxicity, such as weight loss. The low dose should correspond to a no - observed effect level. The oral dosing shall be accomplished by gavage or by administering the encapsulated test substance. If feasible, the same high and low doses should be used for oral and dermal studies. (C) Dermal. (1) Dermal treatment. For dermal treatment, two doses, comparable to the low and high oral doses, shall be dissolved in a suitable vehicle and applied in volumes adequate to deliver comparable doses. The backs of the animals should be lightly shaved with an electric clipper 24 hours before treatment. The test substance shall be applied to the intact shaven skin (approximately 2 cm\2\ for rats, 5 cm\2\ for mini-pigs). The dosed areas shall be protected with a suitable porous covering which is secured in place, and the animals shall be housed separately. (2) Washing efficacy study. Before initiation of the dermal absorption studies, an initial washing efficacy experiment shall be conducted to assess the removal of the applied low dose of the test substance by washing the exposed skin area with soap and water and an appropriate organic solvent. The low dose shall be applied to 4 rats and 4 mini-pigs in accordance with paragraph (c)(2)(ii)(C)(1) of this section. After application (5 to 10 minutes), the treated areas of 2 rats and 2 mini-pigs shall be washed with soap and water and the treated areas of the remaining rats and pigs shall be washed with an appropriate solvent. The amounts of test substance recovered in the washings shall be determined to assess efficacy of its removal by washing. (iii) Dosing and sampling schedule--(A) Rat studies. After administration of the test substance, each rat shall be placed in a metabolic unit to facilitate collection of excreta. For the dermal studies, excreta from the rats shall also be collected during the 6 hour exposure periods. At the end of each collection period, the metabolic units shall be cleaned to recover any excreta that might adhere to them. All studies, except the repeated dosing study, shall be terminated at 7 days or after at least 90 percent of the radioactivity has been recovered in the excreta, whichever occurs first. (1) Intravenous study. Group A shall be dosed once intravenously at the low dose of test substance. (2) Oral study. (i) Group B shall be dosed once per os with the low dose of test substance. (ii) Group C shall be dosed once per os with the high dose of test substance. (3) Dermal studies. Unless precluded by corrosivity, the test substance shall be applied and kept on the skin for a minimum of 6 hours. At the time of removal of the porous covering, the treated area shall be washed with an appropriate solvent to remove any test substance that may be on the skin surface. Both the covering and the washing shall be assayed to recover residual radioactivity. At the termination of the studies, each animal shall be sacrificed and the exposed skin area removed. An appropriate section of the skin shall be solubilized and assayed for radio-activity to ascertain if the skin acts as a reservoir for the test substance. Studies on the dermal absorption of corrosive test substances should be discussed with EPA prior to initiation. (i) Group D shall be dosed once dermally with the low dose of test compound. (ii) Group E shall be dosed once dermally with the high dose of the test substance. (4) Repeated dosing study. Group F shall receive a series of single daily oral low doses of nonradioactive test substance over a period of at least 7 days. Twenty-four hours after the last nonradioactive dose, a single oral low dose of radioactive test substance shall be administered. Following dosing with the radioactive substance, the rats shall be placed in individual metabolic units as described in paragraph (c)(2)(iii) of this section. The study shall be terminated at 7 days after the last dose, or after at least 90 percent of the radioactivity has been recovered in the excreta, whichever occurs first. (B) Mini-Pig studies. For all mini-pig studies, the test groups shall consist of four young adult animals. After administration of the test substance, each mini-pig shall be kept in a metabolic unit to facilitate collection of excreta. At the end of each collection period, the metabolic units are to be cleaned to recover any excreta that might adhere to them. All studies shall be terminated at 7 days, or after at least 90 percent of the radio-activity has been recovered in the excreta, whichever occurs first. (1) Intravenous study. Group G is to be dosed once intravenously at the low dose of the test substance. (2) Dermal studies. Following the experimental guidance described in (c)(2)(iii)(A)(3) of this section: (i) Group H shall be dosed once dermally with the low dose of test substance. (ii) Group I shall be dosed once dermally with the high dose of the test substance. (3) Types of studies--(i) Pharmacokinetics studies--(A) Rat studies. Groups A through F shall be used to determine the kinetics of absorption of the test substance. In the group administered the test substance by intravenous routes, (i.e., Group A), the concentration of radioactivity in blood and excreta shall be measured following administration. In groups administered the test substance by the oral and dermal route (i.e., Groups B, C, D, E and F), the concentration of radioactivity in blood and excreta shall be measured at selected time intervals during and following the exposure period. (B) Mini-Pig studies. Groups G, H, and I shall be used to determine the extent of dermal absorption of the test substance. The amount of radioactivity in excreta shall be determined at selected time intervals. (ii) Metabolism studies--Rat studies. Groups A through F shall be used to determine the metabolism of the test substance. Urine, feces, and expired air shall be collected for identification and quantification of the test substance and metabolites. (4) Measurements--(i) Pharmacokinetics. Four animals from each group shall be used for these purposes. (A) Rat studies--(1) Bioavailability. The levels of radioactivity shall be determined in whole blood, blood plasma or blood serum at 15 and 30 minutes and at 1, 2, 8, 24, 48, and 96 hours after initiation of dosing. (2) Extent of absorption. The total quantities of radioactivity shall be determined for excerta collected daily for 7 days or until at least 90 percent of the radioactivity has been recovered in the excreta. (3) Excretion. The quantities of radioactivity eliminated in the urine, feces, and expired air shall be determined separately at appropriate time intervals. The collection of carbon dioxide may be discontinued when less than one percent of the dose is found to be exhaled as radioactive carbon dioxide in 24 hours. (4) Tissue distribution. At the termination of each study, the quantities of radioactivity in blood and in various tissues, including bone, brain, fat, gastrointestinal tract, gonads, heart, kidney, liver, lungs, muscle, skin, and residual carcass of each animal shall be determined. (5) Changes in pharmacokinetics. Results of pharmacokinetics measurements (i.e., bioavailability and extent of absorption, tissue distribution, and excretion) obtained in rats receiving the single low oral dose of the test substance (Groups B and C) shall be compared to the corresponding results obtained in rats receiving repeated oral doses of the test substance (Group F). (B) Mini-Pig studies--Extent of absorption. The total quantities of radioactivity shall be determined for excreta daily for 7 days or until at least 90 percent of the test substance has been excreted. (ii) Metabolism. Four animals from each group shall be used for these purposes. (A) Rat studies--(1) Biotransformation. Appropriate qualitative and quantitative methods shall be used to assay urine, feces, and expired air collected from rats. Efforts shall be made to identify any metabolite which comprises 5 percent or more of the administered dose and the major radioactive components of blood. (2) Changes in biotransformation. Appropriate qualitative and quantitative assay methodology shall be used to compare the composition of radioactive compounds in excreta from rats receiving a single oral dose (Groups B and C) with those in the excreta from rats receiving repeated oral doses (Group H). (d) Data and reporting. The final test report shall include the following: (1) Presentation of results. Numerical data shall be summarized in tabular form. Pharmacokinetic data shall also be presented in graphical form. Qualitative observations shall also be reported. (2) Evaluation of results. All quantitative results shall be evaluated by an appropriate statistical method. (3) Reporting results. In addition to the reporting requirements as specified in 40 CFR part 792, the following specific information shall be reported: (i) Species and strains of laboratory animals. (ii) Chemical characterization of the test substance, including: (A) For the radioactive test substances, information on the site(s) and degree of radiolabeling, including type of label, specific activity, chemical purity, and radiochemical purity. (B) For the nonradioactive compound, information on chemical purity. (C) Results of chromatography. (iii) A full description of the sensitivity, precision, and accuracy of all procedures used to generate the data. (iv) Percent of absorption of test substance after oral and dermal exposures to rats and dermal exposure to mini-pigs. (v) Quantity and percent recovery of radioactivity in feces, urine, expired air, and blood. In dermal studies on rats and mini-pigs, include recovery data for skin, skin washings, and residual radioactivity in the covering as well as results of the washing efficacy study. (vi) Tissue distribution reported as quantity of radioactivity in blood and in various tissues, including bone, brain, fat, gastrointestinal tract, gonads, heart, kidney, liver, lung, muscle, skin and in residual carcass of rats. (vii) Materials balance developed from each study involving the assay of body tissues and excreta. (viii) Biotransformation pathways and quantities of test substance and metabolites in excreta collected after administering single high and low doses to rats. (ix) Biotransformation pathways and quantities of the test substance and metabolites in excreta collected after administering repeated low doses to rats. (x) Pharmacokinetics model(s) developed from the experimental data. [54 FR 33411, Aug. 14, 1989; 54 FR 49844, Dec. 1, 1989; 55 FR 25392, June 21, 1990] 795.230 Oral and inhalation pharmacokinetic test. (a) Purpose. The purpose of these studies is to determine: (1) Bioavailability of test substance after oral and inhalation exposure. (2) Whether or not the biotransformation of the test substance is qualitatively and quantitatively the same after oral and inhalation exposure. (3) Whether or not the biotransformation of the test substance is changed qualitatively or quantitatively by repeated dosing. (b) Definitions. Bioavailability refers to the rate and extent to which an administered chemical substance compound is absorbed, i.e., reaches the systemic circulation. (c) Test procedures--(1) Animal selection--(i) Species. The preferred species is the rat for which extensive data on the toxicity and carcinogenicity of numerous chemical substances are available. (ii) Animals. Adult male and female Fischer 344 rats shall be used. The rats shall be 7 to 9 weeks old. Prior to testing, the animals are selected at random for each group. Animals showing signs of ill health shall not be used. (iii) Animal care. Animals shall be housed in environmentally controlled rooms with 10 to 15 air changes per hour. The rooms shall be maintained at a temperature of 24 ÷2 +C and humidity 50 ÷ 10 percent with a 12-hour light/dark cycle per day. The rats shall be isolated for at least 7 days prior to use, and their health status shall then be evaluated. The animals shall be acclimated to the experimental environment for a minimum of 48 hours prior to treatment. Certified feed and water shall be provided ad libitum. (iv) Numbers.--(A) At least 8 animals (4 males and 4 females) shall be used at each dose level. (B) Females shall be nulliparous and nonpregnant. (2) Administration of the test substance--(i) Test substance. The test substance shall be at least 99 percent pure. The studies require the use of both nonradioactive and \14\C-labeled test substance. Both preparations are needed to investigate the provisions of paragraph (a)(2) of this section. The use of \14\C-test substance is recommended for the provisions in paragraphs (a) (1), (2), and (3) of this section in order to facilitate the work, improve the reliability of quantitative determinations, and increase the probability of observing previously unidentified metabolites. (ii) Dosage and treatment--(A) Oral study. At least two doses shall be used in the study, a ``low'' and ``high'' dose. When administered orally, the ``high'' dose should induce some overt toxicity such as weight loss. The ``low'' dose shall not induce observable effects attributable to the test substance. Oral dosing shall be performed by gavage using an appropriate vehicle. (B) Inhalation study. Three concentrations shall be used in the study. Upon exposure, the two higher concentrations should ideally induce some overt symptoms of toxicity, although the intermediate concentration may be excluded from this condition. The lowest concentration shall not induce observable effects attributable to the test substance. (iii) Determination of bioavailability--(A) Oral studies. (1) Group A (a minimum of 8 animals, 4 males and 4 females) shall be dosed once per os with the low dose of \14\C-labeled test substance. (2) Group B (a minimum of 8 animals, 4 males and 4 females) shall be dosed once per os with the high dose of \14\C-labeled test substance. (B) Inhalation studies. (1) Group C (a minimum of 8 animals, 4 males and 4 females) shall be exposed (6 hours) to a mixture of non-radioactive test substance in air at the prescribed low test substance concentration. (2) Group D (a minimum of 8 animals, 4 males and 4 females) shall be exposed (6 hours) to a mixture of non-radioactive test substance in air at the prescribed intermediate test substance concentration. (3) Group E (a minimum of 8 animals, 4 males and 4 females) shall be exposed (6 hours) to a mixture of non-radioactive test substance in air at the prescribed high concentration. (4) Group F (a minimum of 8 animals, 4 males and 4 females) shall be exposed (6 hours) to a mixture of \14\C-labeled test substance in air at the prescribed low test substance concentration. (5) Group G (a minimum of 8 animals, 4 males and 4 females) shall be exposed (6 hours) to a mixture of \14\C - labeled test substance in air at the prescribed intermediate test substance concentration. (6) Group H (a minimum of 8 animals, 4 males and 4 females) shall be exposed (6 hours) to a mixture of \14\C-labeled test substance in air at the prescribed high test substance concentration. (C) Collection of excreta. After oral administration (Groups A and B) and inhalation exposure (Groups F through H) the rats shall be placed in individual metabolic cages and excreta (urine, feces and expired air) shall be collected from 0 to 24 hours and then from 24 to 48 hours post-treatment, or until 90 percent of the dose has been excreted, whichever occurs first. (D) Kinetic studies. Groups C through E shall be used to determine the concentration of the test substance in blood at 0, 5, 10, 15, and 30 minutes, and at 1, 2, 4, 8, 16, 24, and 48 hours after initiation of inhalation exposure. (E) Repeated dosing study. Rats (a minimum of 8 animals, 4 males and 4 females) shall receive a series of single daily oral doses of non-radioactive test substance over a period of at least 7 days, followed at 24 hours after the last dose by a single oral dose of \14\C-labeled test substance. Each dose shall be at the low-dose level. Urine shall be collected from 0 to 24 hours and then 24 to 48 hours after administering the \1\\4\C-labeled test substance. (3) Observation of animals--(i) Bioavailability--(A) Blood levels. The levels of total \14\C-label shall be determined in whole blood, blood plasma, or blood serum of each rat at 0, 4, 8, 16, 24, and 48 hours after dosing rats in Groups A - B and F - H. (B) Expired air, urinary and fecal excretion. The quantities of total \14\C-label eliminated in expired air, urine, and feces by each rat in Groups A and B and F through H shall be determined in collections made from 0 to 24 hours and then 24 to 48 hours after dosing and, if necessary, daily thereafter until at least 90 percent of the dose has been excreted or until 7 days after dosing, whichever occurs first. (C) Tissue distribution. The concentration and quantity of \14\C-label in tissue and organs shall be determined at the time of sacrifice for each rat in Groups A and B, F through H, and the repeated-dosing group. (ii) Biotransformation after oral and inhalation exposure. Appropriate qualitative and quantitative methods shall be used to assay urine specimens collected from each rat in Groups A and B and F through H. Suitable enzymatic steps should be used to distinguish, characterize, and quantify conjugated and unconjugated metabolites of the test substance. (iii) Change(s) in biotransformation. Appropriate qualitative and quantitative assay methodologies shall be used to compare the composition of \14\C-labeled components of urine collected from 0 to 24 and then from 24 to 48 hours after dosing rat Group A with those components in the urine collected over the same intervals after administering the radioactive dose in the repeated dosing study. (d) Data and reporting--(1) Treatment of results. Data should be summarized in tabular form. (2) Evaluation of results. All observed results shall be evaluated by an appropriate statistical method. (3) Test report. In addition to the reporting requirements as specified in the EPA Good Laboratory Practice Standards (subpart J, part 792 of this chapter) the following specific information should be reported: (i) Labeling site of the test substance. (ii) A full description of the sensitivity and precision of all procedures used to produce the data. (iii) Quantity of isotope, together with percent recovery of the administered dose in feces, urine, expired air, and blood for both routes of administration. (iv) Quantity and distribution of \14\C-test substance in bone, brain, fat, gonads, heart, kidney, liver, lung, muscle, spleen, tissue which displayed pathology, and residual carcass. (v) Biotransformation pathways and quantities of test substance and its metabolites in urine, feces, and expired air collected after oral administration (single low and high doses) and inhalation exposure (low, intermediate, and high concentrations). (vi) Biotransformation pathways and quantities of the test substance and its metabolites in urine collected after repeated administration of test substance to rats. (vii) Pharmacokinetic model(s), if any, developed from the experimental data. (4) Counting efficiency. Data should be made available to the Agency upon request. [52 FR 37143, Oct. 5, 1987] 795.231 Pharmacokinetics of isopropanal. (a) Purpose. The purposes of these studies are to: (1) Ascertain whether the pharmacokinetics and metabolism of the ``test substance'' are similar after oral and inhalation administration. (2) Determine bioavailability of the test substance after oral and inhalation administration. (3) Examine the effects of repeated dosing on the pharmacokinetics and metabolism of the test substance. (b) Definitions. (1) ``Bioavailability'' refers to the rate and relative amount of administered test substance which reaches the systemic circulation. (2) ``Metabolism'' means the study of the sum of the processes by which a particular substance is handled in the body, and includes absorption, tissue distribution, biotransformation, and excretion. (3) ``Pharmacokinetics'' means the study of the rates of absorption, tissue distribution, biotransformation, and excretion. (c) Test procedures--(1) Animal selection--(i) Species. The rat shall be used because it has been used extensively for metabolic and toxicological studies. (ii) Test animals. For pharmacokinetics testing, adult male and female rats (Fischer 344 or strain used for major toxicity testing), 7 to 9 weeks of age, shall be used. The animals should be purchased from a reputable dealer and shall be identified upon arrival at the testing laboratory. The animals shall be selected at random for the testing groups and any animal showing signs of ill health shall not be used. In all studies, unless otherwise specified, each test group shall contain at least four animals of each sex for a total of at least eight animals. (iii) Animal care. (A) Animal care and housing should be in accordance with DHEW Publication No. (NIH)-85-23, 1985, entitled ``Guidelines for the Care and Use of Laboratory Animals.'' (B) The animals should be housed in environmentally controlled rooms with at least 10 air changes per hour. The rooms shall be maintained at a temperature of 22÷2 +C and humidity of 50÷20 percent with a 12-hour light/dark cycle per day. The animals shall be kept in a quarantine facility for at least 7 days prior to use and shall be acclimated to the experimental environment for a minimum of 48 hours prior to treatment. (C) During the acclimatization period, the animals should be housed in suitable cages. All animals shall be provided with certified feed and tap water ad libitum. (2) Administration of test substance--(i) Test substance. The use of radioactive test substance is required for all materials balance and metabolite identification requirements of the study. Ideally, the purity of both radioactive and nonradioactive test substance should be greater than 99 percent. The radioactive and nonradioactive substances shall be chromatographed separately and together to establish purity and identity. If the purity is less than 99 percent or if the chromatograms differ significantly, EPA should be consulted. (ii) Dosage and treatment--(A) Intravenous. The low dose of test substance, in an appropriate vehicle, shall be administered intravenously to four rats of each sex. (B) Oral. Two doses of test substance shall be used in the oral portion of the study, a low dose and a high dose. The high dose should ideally induce some overt toxicity, such as weight loss. The low dose level should correspond to a no-observed effect level. The oral dosing shall be accomplished by gavage or by administering an encapsulated test substance. If feasible, the same high and low doses should be used for oral and dermal studies. (C) Inhalation. Two concentrations of the test substance shall be used in this portion of the study, a low concentration and a high concentration. The high concentration should ideally induce some overt toxicity, while the low concentration should correspond to a no observed level. Inhalation treatment should be conducted using a ``nose-cone'' or ``head only'' apparatus to prevent ingestion of the test substance through ``grooming''. (iii) Dosing and sampling schedule. After administration of the test substance, each rat shall be placed in a separate metabolic unit to facilitate collection of excreta. For the inhalation studies, excreta from the rats shall also be collected during the exposure periods. At the end of each collection period, the metabolic units shall be cleaned to recover any excreta that might adhere to the cages. All studies, except the repeated dose study, shall be terminated at 7 days, or after at least 90 percent of the radioactivity has been recovered in the excreta, whichever occurs first. (A) Intravenous study. Group A shall be dosed once intravenousely at the low dose of test substance. (B) Oral studies. (1) Group B shall be dosed once per os with the low dose of the test substance. (2) Group C shall be dosed once per os with the high dose of the test substance. (C) Inhalation studies. A single 6-hour exposure period shall be used for each group. (1) Group D shall be exposed to a mixture of the test substance in air at the low concentration. (2) Group E shall be exposed to a mixture of test substance in air at the high concentration. (D) Repeated dosing study. Group F shall receive a series of single daily oral low doses of nonradioactive test substance over a period of at least 7 consecutive days. Twenty four hours after the last nonradioactive dose, a single oral low dose of radioactive test substance shall be administered. Following dosing with radioactive substance, the rats shall be placed in individual metabolic units as described in paragraph (c)(2)(iii) of this section. The study shall be terminated 7 days after the last dose, or after at least 90 percent of the radioactivity has been recovered in the excreta, whichever occurs first. (3) Types of studies--(i) Pharmacokinetics studies. Groups A through F shall be used to determine the kinetics of absorption of the test substance. In groups administered the substance by intravenous or oral routes, (i.e., Groups A, B, C, F), the concentration of radioactivity in blood and excreta including expired air shall be measured following administration. In groups administered the substance by the inhalation route (i.e., Groups D and E), the concentration of radioactivity in blood shall be measured at selected time intervals during and following the exposure period. In the groups administered the substance by inhalation (i.e., Groups D and E), the concentration of radioactivity in excreta (including expired air) shall be measured at selected time intervals following the exposure period. In addition, in the groups administered the substance by inhalation, the concentration of test substance in inspired air shall be measured at selected time intervals during the exposure period. (ii) Metabolism studies. Groups A through F shall be used to determine the metabolism of the test substance. Excreta (urine, feces, and expired air) shall be collected for identification and quantification of test substance and metabolites. (4) Measurements--(i) Pharmacokinetics. Four animals from each group shall be used for these purposes. (A) Bioavailability. The levels of radioactivity shall be determined in whole blood, blood plasma or blood serum at 15 minutes, 30 minutes, 1, 2, 3, 6, 9, and 18 hours after dosing; and at 30 minutes, 3, 6, 6.5, 7, 8, 9, 12, and 18 hours after initation of inhalation exposure. (B) Extent of absorption. The total quantities of radioactivity shall be determined for excreta collected daily for 7 days, or after at least 90 percent of the radioactivity has been recovered in the excreta, whichever occurs first. (C) Excretion. The quantities of radioactivity eliminated in the urine, feces, and expired air shall be determined separately at appropriate time intervals. The collection of the intact test substance or its metabolites, including carbon dioxide, may be discontinued when less than 1 percent of the administered dose is found to be exhaled as radioactive carbon dioxide in 24 hours. (D) Tissue distribution. At the termination of each study, the quantities of radioactivity in blood and in various tissues, including bone, brain, fat, gastrointestinal tract, gonads, heart, kidney, liver, lungs, muscle, skin, spleen, and residual carcass of each animal shall be determined. (E) Changes in pharmacokinetics. Results of pharmacokinetics measurements (i.e., biotransformation, extent of absorption, tissue distribution, and excretion) obtained in rats receiving the single low oral dose of test substance (Group B) shall be compared to the corresponding results obtained in rats receiving repeated oral doses of test substance (Group F). (F) Biotransformation. Appropriate qualitative and quantitative methods shall be used to assay urine, feces, and expired air collected from rats. Efforts shall be made to identify any metabolite which comprises 5 percent or more of the dose eliminated. (G) Changes in biotransformation. Appropriate qualitative and quantitative assay methodology shall be used to compare the composition of radioactive substances in excreta from the rats receiving a single oral dose (Groups B and C) with those in the excreta from rats receiving repeated oral doses (Group F). (ii) [Reserved] (d) Data and reporting. The final test report shall include the following: (1) Presentation of results. Numerical data shall be summarized in tabular form. Pharmacokinetics data shall also be presented in graphical form. Qualitative observations shall also be reported. (2) Evaluation of results. All quantitative results shall be evaluated by an appropriate statistical method. (3) Reporting results. In addition to the reporting requirements as specified in the EPA Good Laboratory Practice Standards (40 CFR 792.185), the following specific information shall be reported: (i) Species and strains of laboratory animals. (ii) Chemical characterization of the test substance, including: (A) For the radioactive test substance, information on the site(s) and degree of radiolabeling, including type of label, specific activity, chemical purity, and radiochemical purity. (B) For the nonradioactive substance, information on chemical purity. (C) Results of chromatography. (iii) A full description of the sensitivity, precision, and accuracy of all procedures used to generate the data. (iv) Extent of absorption of the test substance as indicated by: percent absorption of the administered oral dose; and total body burden after inhalation exposure. (v) Quantity and percent recovery of radioactivity in feces, urine, expired air, and blood. (vi) Tissue distribution reported as quantity of radioactivity in blood and in various tissues, including bone, brain, fat, gastrointestinal tract, gonads, heart, kidney, liver, lung, muscle, skin, spleen and in residual carcass of each rat. (vii) Biotransformation pathways and quantities of the test substance and metabolites in excreta collected after administering single high and low doses to rats. (viii) Biotransformation pathways and quantities of the test substance and metabolites in excreta collected after administering repeated low doses to rats. (ix) Pharmacokinetics model(s) developed from the experimental data. [54 FR 43261, Oct. 23, 1989] 795.232 Inhalation and dermal pharmacokinetics of commercial hexane. (a) Purposes. The purposes of these studies are to: (1) Determine the bioavailability of the test substances after dermal and inhalation administration. (2) Compare the pharmacokinetics and metabolism of the test substances after intravenous, dermal, and inhalation administration. (3) Examine the effects of repeated doses on the pharmacokinetics and metabolism of the test substances. (b) Definitions. (1) ``Bioavailability'' refers to the relative amount of administered test substance which reaches the systemic circulation and the rate at which this process occurs. (2) ``Metabolism'' means the sum of the enzymatic and nonenzymatic processes by which a particular substance is handled in the body. (3) ``Pharmacokinetics'' means the study of the rates of absorption, tissue distribution, biotransformation, and excretion. (4) ``Low dose'' should correspond to 1/10 of the high dose. (5) ``High dose'' shall not exceed the lower explosive limit (LEL) and ideally should induce minimal toxicity. (6) ``Test substance'' refers to the unlabeled and both radiolabeled mixtures (14C-n-hexane and 14C-methylcyclopentane) of commercial hexane used in the testing. (c) Test procedures--(1) Animal selection--(i) Species. The rat shall be used for pharmacokinetics testing because it has been used extensively for metabolic and toxicological studies. (ii) Test animals. Adult male and female rats shall be used for testing. The rats shall be 7 to 9 weeks old and their weight range should be comparable from group to group. The animals shall be purchased from a reputable dealer and shall be permanently identified upon arrival. The animals shall be selected at random for the testing groups, and any animal showing signs of ill health shall not be used. (iii) Animal care. (A) Animal care and housing shall be in accordance with DHHS/PHS NIH Publication No. 86 - 23, 1985, ``Guidelines for the Care and Use of Laboratory Animals.'' (B) The animals shall be housed in environmentally controlled rooms with at least 10 air changes per hour. The rooms shall be maintained at a temperature of 18 to 26 degrees centigrade and humidity of 40 to 70 percent with a 12 - hour light/dark cycle per day. The animal subjects shall be kept in a quarantine facility for at least 7 days prior to use, and shall be acclimated to the experimental environment for a minimum of 48 hours prior to treatment. (C) During the acclimatization period, the rats shall be housed in suitable cages. All animals shall be provided with certified feed and tap water ad libitum. (2) Administration of test substances--(i) Test substances. The study will require the use of both radiolabeled and unlabeled test substances. All unlabeled commercial hexane shall be from the same lot number. Two kinds of radiolabeled test substances will be tested. 14C-n-hexane shall be the only radiolabeled component of one, and 14C-MCP shall be the only radiolabeled component of the other test substance. The use of both radiolabeled test substances is required for all pharmacokinetics and metabolism studies described in this rule, except for the bioavailability measurements required in (c)(4)(i)(A) of this section. The bioavailability measurements need only be conducted with the test substance containing 14C-n-hexane or an unlabeled test substance may be used if it can be demonstrated that the analytical sensitivity of the method used with the unlabeled test substance is equal to or greater than the sensitivity which could be obtained with the radiolabeled test substance. If an unlabeled test substance is used for bioavailability measurements, these measurements shall be extended to include relevant metabolites of n-hexane. These test substances shall contain at least 40 liquid volume percent but no more than 55 liquid volume percent n-hexane and no less than 10 liquid volume percent methylcyclopentane (MCP) and otherwise conform to the specifications prescribed in the American Society for Testing and Materials Designation D 1836 - 83 (ASTM D 1836), ``Standard Specification for Commercial Hexanes'', published in the 1986 Annual Book of ASTM Standards: Petroleum Products and Lubricants, ASTM D 1836 - 83, pp. 966 - 967, 1986, which is incorporated by reference in accordance with 5 U.S.C. 552(a). ASTM D 1836 - 83 is available for public inspection at the Office of the Federal Register, Rm. 8301, 11th and L St., NW., Washington, DC 20408, and copies may be obtained from the EPA, TSCA Public Docket Office, Rm. NE G - 004, 401 M St., SW., Washington, DC 20460. This incorporation by reference was approved by the Director of the Office of the Federal Register in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. This material is incorporated as it exists on the date of approval, and a notice of any change in this material will be published in the Federal Register. (ii) Dosage and treatment--(A) Intravenous. An appropriate dose of the test substance shall be administered intravenously. The intravenous data obtained in this portion of the study shall be suitable for the determination of absorption, distribution, and excretion parameters of the test substance. Factors that should be considered in the selection of the intravenous doses are: The acute toxicity of the test substance, the availability of a suitable vehicle (if saline is unsuitable) and the solubility of the test substance in the vehicle. (B) Inhalation. Two concentrations of each test substance shall be used in this portion of the study, a low concentration and a high concentration. The high concentration should induce minimal toxicity, but shall not exceed the lower explosive limit (LEL). The low concentration shall correspond to 1/10 of the high concentration. Inhalation treatment shall be conducted using a ``nose-cone'' or ``head only'' apparatus to reduce ingestion of the test substance through ``grooming'' or dermal absorption. (C) Dermal. Dermal absorption studies should be conducted by the methodology of Susten, A.S., Dames, B.L. and Niemeier, R.W., ``In vivo percutaneous absorption studies of volatile solvents in hairless mice. I. Description of a skin depot'', In: Journal of Applied Toxicology 6:43 - 46, (1986), or by some other suitable method because the test substances have significant volatility. The high and low doses shall be tested in rats. (iii) Dosing and sampling schedule. Each experimental group shall contain at least four animals of each sex. After administration of the test substance, each rat shall be placed in an individual metabolic unit for collection of urine, feces, and expired air. For the dermal studies, excreta from the rats shall also be collected during the exposure periods. At the end of each collection period, the metabolic units shall be cleaned to recover any excreta that might adhere to the units. All studies, except the repeated dose studies, shall be terminated at 7 days, or after at least 90 percent of the administered radioactivity has been recovered in the excreta, whichever occurs first. All studies described below shall be conducted separately with each radiolabeled test substance. (A) Intravenous study. Group A shall be given a single intravenous dose of the radiolabeled test substance to result in a level of commercial hexane in the blood that approximates the level from the other routes of exposure so that the data can be used to determine absorption and excretion parameters. (B) Inhalation studies. A single 6 - hour exposure period shall be used for each group. (1) Group B shall be exposed to a mixture of the radiolabeled test substance in air at the low concentration. (2) Group C shall be exposed to a mixture of the radiolabeled test substance in air at the high concentration. (C) Dermal studies. The test substance shall be applied and kept on the skin for a minimum of 6 hours. The covering apparatus components shall be assayed to recover residual radioactivity. At the termination of the studies, each animal shall be sacrificed and the exposed skin area removed. An appropriate section of the skin shall be solubilized and assayed for radioactivity to ascertain whether the skin acts as a reservoir for the test substance. (1) Group D shall be given one dermal, low dose of the radiolabeled test substance. (2) Group E shall be given one dermal, high dose of the radiolabeled test substance. (D) Repeated dosing study. Group F shall receive a series of single daily 6 - hour inhalation exposures to unlabeled test substance at the low dose over a period of at least 7 days. A single 6 - hour inhalation exposure to the radiolabeled test substance at the low dose shall be administered 24 hours after the last unlabeled exposure. Following administration of the radiolabeled substance, the rats shall be placed in individual metabolic units and excreta collected. The study shall be terminated 7 days after the last exposure, or after at least 90 percent of the radioactivity has been recovered in the excreta, whichever occurs first. (3) Types of studies--(i) Pharmacokinetics studies. Groups A through F shall be used to determine the kinetics of absorption of the test substance. In animal subjects administered the test substance intravenously (i.e., Group A), the concentration of test substance in blood and excreta shall be measured following administration. In animal subjects administered the test substance by the inhalation and dermal routes (i.e., Groups B through F), the concentration of test substance in blood shall be measured at selected time intervals during and following the exposure period. In animal subjects administered the test substance by the inhalation route (i.e., Groups B, C, and F) the concentration of test substance in excreta shall be measured following exposure. In animal subjects administered the test substance by the dermal route (i.e., Groups D and E) the concentration of test substance in excreta shall be measured during and following exposure. These measurements allow calculation of uptake, half lives, and clearance. In addition, in the groups administered the test substance by inhalation (i.e., Groups B, C, and F), the concentration of test substance in the exposure chamber air shall be measured at selected time intervals during the exposure period. (ii) Metabolism studies. Groups A through F shall be used to determine the metabolism of the test substance. Excreta (urine, feces, and expired air) shall be collected for identification and measurement of the quantities of test substance and metabolites. (4) Measurements--(i) Pharmacokinetics. At least four animals from each group shall be used for these purposes. (A) Bioavailability. The levels of test substance and relevant metabolites, as appropriate, shall be determined in whole blood, blood plasma or blood serum at appropriate intervals after initiation of intravenous, dermal, and inhalation exposure. The sampling intervals should be compatible with the exposure route under study. The determinations need only be done on animals administered the test substance containing 14C-n-hexane or, if the analytical sensitivity is equal or greater, unlabeled test substance may be used. (B) Extent of absorption. The total quantities of radioactivity shall be determined for excreta collected daily for 7 days, or until at least 90 percent of theradioactivity has been recovered in the excreta, whichever occurs first. (C) Excretion. The quantities of radioactivity eliminated in the urine, feces, and expired air shall be determined separately at time intervals that provide accurate measurement of clearance and excretory rates. The collection of carbon dioxide may be discontinued when less than one percent of the dose is found to be exhaled as radioactive carbon dioxide in 24 hours. (D) Tissue distribution. At the termination of each study, the quantities of radioactivity shall be determined in blood and in various tissues, including bone, brain, fat, gastrointestinal tract, gonads, heart, kidney, liver, lungs, muscle, skin, spleen, thymus, and residual carcass of each animal. (E) Change in pharmacokinetics. Results of pharmacokinetics measurements (i.e., biotransformation, extent of absorption, tissue distribution, and excretion) obtained in rats receiving the single inhalation exposure to the low dose of the test substance (Group B) shall be compared to the corresponding results obtained in rats receiving repeated inhalation exposures to the low dose of the test substance (Group F). (ii) Metabolism. At least four animals from each group shall be used for these purposes. (A) Biotransformation. Appropriate qualitative and quantitative methods shall be used to assay urine, feces, and expired air collected from rats. Efforts shall be made to identify any metabolite which comprises 5 percent or more of the dose administered. (B) Changes in biotransformation. Appropriate qualitative and quantitative assay methods shall be used to compare the composition of radioactive compounds in excreta from rats receiving a single inhalation exposure (Groups B and C) with that from rats receiving repeated inhalation exposures (Group F). (d) Data and reporting. The final test report shall include the following: (1) Presentation of results. Numerical data shall be summarized in tabular form. Pharmacokinetics data shall also be presented in graphical form. Qualitative observations shall also be reported. (2) Evaluation of results. All data shall be evaluated by appropriate statistical methods. (3) Reporting results. In addition to the reporting requirements as specified in 40 CFR part 792, the following information shall be reported. (i) Strain of laboratory animals. (ii) Chemical characterization of the test substances, including: (A) For the radiolabeled test substances, information on the sites and degree of radiolabeling, including type of label, specific activity, chemical purity prior to mixing with the unlabeled hexane mixture, and radiochemical purity. (B) For the unlabeled test substance, information on lot number and the percentage of MCP and n-hexane. (C) Results of chromatography. (iii) A full description of the sensitivity, precision, and accuracy of all procedures used to obtain the data. (iv) Percent and rate of absorption of the test substance after inhalation and dermal exposures. (v) Quantity and percent recovery of radioactivity in feces, urine, expired air, and blood. For dermal studies, include recovery data for skin and residual radioactivity in the covering apparatus. (vi) Tissue distribution reported as quantity of radioactivity in blood, in various tissues including bone, brain, fat, gastrointestinal tract, gonads, heart, kidney, liver, lung, muscle, skin, spleen, thymus, and in residual carcass. (vii) Biotransformation pathways, to the extent possible, and quantities of the test substances and metabolites in excreta collected after administering single high and low doses. (viii) Biotransformation pathways, to the extent possible, and quantities of test substances and metabolites in excreta collected after administering repeated low doses. (ix) Pharmacokinetics models to the extent they can be developed from the experimental data. (Approved by the Office of Management and Budget under control number 2070 - 0033) [55 FR 632, Jan. 8, 1990] 795.235 Toxicokinetic Test. (a) Purpose. These studies are designed to: (1) Determine the bioavailability of the test substance after dermal or oral treatment. (2) Ascertain whether the metabolites of the test substance are similar after dermal (assuming significant penetration) and oral administration. (3) Examine the effects of a multiple dosing regimen on the metabolism of the test substance after per os administration. (b) Definition of scope of study. Absorption toxicokinetics refers to the bioavailability, i.e., the rate and extent of absorption of the test substance, and metabolism and excretion rates of the test substance after absorption. (c) Test procedures--(1) Animal selection--(i) Species. The rat is the animal species of choice since it has been used extensively for absorption, metabolism, and toxicological studies. (ii) Rat strain. Adult male and female Fischer 344 rats shall be used. At 7 to 9 weeks of age, the males should weigh 125 to 175 g and the females 110 to 150 g. The rats shall be purchased from a reputable dealer and identified with ear tags upon arrival. The animals shall be randomly selected for the testing groups, and no unhealthy animal is to be used for experimentation. (iii) Animal care. (A) Animal care and housing should be in accordance with Department of Health, Education and Welfare Publication No. (NIH) - 78 - 23, 1978. ``Guidelines for the Care and Use of Laboratory Animals,'' or its equivalent. (B) The animals shall be housed in environmentally controlled rooms with 10 to 15 air changes per hour. The rooms shall be maintained at a temperature of 25÷2 +C and humidity of 50÷10 percent with a 12-hour light/dark cycle per day. The rats shall be kept in a quarantine facility for at least 7 days prior to use. (C) During the acclimatization period, the rats shall be housed in polycarbonate cages on hardwood chip bedding. All animals shall be provided with certified feed and tap water ad libitum. (iv) Number of animals. There shall be at least four animals of each sex in each experimental group. (2) Administration of test substance--(i) Test substance. Test substance of at least 99 percent purity, commercially available, should be used as the test substance. Since both nonradioactive and radioactive (uniformly \14\C-labelled) test substances are to be used, they should be chromatographed separately and analyzed together, to ascertain purity and identity. The use of \14\C-labelled test substance, diluted with unlabeled test substance, is required for all of the studies under this section, unless otherwise specified, as it will greatly increase the reliability and sensitivity of the quantitative assays and facilitate the identification of metabolites. (ii) Dosage and treatment. (A) Two doses shall be used in studies under this section, a ``low'' dose and a ``high'' dose. When administered orally, the ``high'' dose level should ideally induce some overt toxicity, such as weight loss. The ``low'' dose level should not induce observable effects attributable to the test substance. If feasible, the same ``high'' and ``low'' doses should be administered orally and dermally. (B) Oral dosing shall be accomplished by gavage after dissolving the test substance in a suitable vehicle. For dermal treatment, the doses shall be administered in a suitable solvent and applied at a volume adequate to deliver the prescribed doses. The backs of the rats should be shaved with an electric clipper one day before treatment. The dose should be applied with a disposable micropipette on a specific area (2 cm\2\ for rats) on the shaven skin. The dosed areas shall be occluded with an aluminum foil patch which is secured in place with adhesive tape. (iii) Determination of test substance kinetics. Each experimental group shall contain at least four rats of each sex for a total of eight rats. (A) Oral studies. (1) Group A shall be dosed once per os with the low dose of the test substance. (2) Group B shall be dosed once per os with the high dose of the test substance. (3) For the oral studies, the rats shall be placed in individual metabolic cages to facilitate collection of urine and feces at 8, 24, 48, 72, and 96 hours following administration. The cages shall be cleaned at each time period to collect any metabolites that might adhere to the metabolic cages. (B) Dermal Studies. (1) Group C shall be dosed once dermally with the low dose of test substance. (2) Group D shall be dosed once dermally with the high dose of test substance. (3)(i) For the dermal studies, the test substance shall be applied for 24 hours. Immediately after application, each animal shall be placed in a separate metabolic cage for excreta collection. At the time of removal of the aluminum foil, the occluded area shall be washed with an appropriate solvent (see below), to remove any test substance that may be on the skin surface and the wash solvent assayed for the amount of test substance recovered. At the termination of the experiments, each animal shall be sacrificed and the exposed skin area removed. The skin (or an appropriate section) shall be solubilized and assayed for the test substance and its metabolites. (ii) Before initiation of the dermal studies, an initial washing efficiency experiment shall be conducted to assess the removal of the applied test substance by washing the exposed skin area with soap and water or organic solvents. Four rats, two of each sex, shall be lightly anesthetized and then test substance applied to a specified area. After application (5 to 10 minutes), the areas should be washed with soap and water (two rats) or ethanol and water (two rats). The amount recovered shall be determined to assess efficacy of test substance removal by washing of the skin. (C) Repeated dosing study group E. Four rats (two of each sex) shall receive a series of single daily oral doses of nonradioactive test substance over a period of at least 14 days, followed at 24 hours after the last dose by a single oral dose of \14\C-labelled test substance. Each dose shall be at the low dose level. (3) Observation of animals--(i) Bioavailability--(A) Blood levels. The levels of \14\C shall be determined in whole blood, blood plasma, or blood serum at appropriate intervals from 1 to 96 hours after dosing rats in Groups A through E. Four rats (two of each sex) of each group shall be used for this purpose. (B) Urinary and fecal excretion. The quantities of \14\C excreted in the urine and feces by rats in Groups A through E shall be determined at 8 hours, 24 hours, 48 hours, 72 hours, and 96 hours after dosing, and if necessary, daily thereafter until at least 90 percent of the applied dose has been excreted or until 7 days after dosing (whichever occurs first). Four animals (two of each sex) shall be used for these analyses. (ii) Biotransformation after oral and dermal dosing. Appropriate qualitative and quantitative methods shall be used to assay the test substance and metabolites in the urine and fecal specimens collected from rat Groups A through D. (iii) Changes in Biotransformation. Appropriate qualitative and quantitative assay methodology shall be used to compare the composition of \14\C-labelled compounds in excreta collected at 14 and 48 hours after dosing rat Group A with those in the excreta collected at 24 and 48 hours after the \14\C-labelled test substance dose in the repeated dose study (Group E). (d) Data and reporting--(1) Treatment of results. Data should be summarized in tabular form. (2) Evaluation of results. All observed results, quantitative or incidental, should be evaluated by an appropriate statistical method. (3) Test report. In addition to the reporting requirements specified in the EPA Good Laboratory Practice Standards (40 CFR part 792, subpart J) the following specific information shall be reported: (i) Specie(s) and strain(s) of laboratory animals. (ii) Information on the degree (i.e., specific activity for a radiolabel) and site(s) of labeling of the test substance; (iii) A full description of the sensitivity and precision of all procedures used to produce the data. (iv) Percent absorption by oral and dermal routes of rats administered \14\C-test substance. (v) Quantity of isotope, together with percent recovery of administered dose in feces, urine, blood, and skin and skin washings (dermal study only for last two portions). (vi) Quantity and distribution of \14\C-labelled test substance in various tissues, including bone, brain, fat, gonads, heart, kidney, liver, lung, muscle, spleen, and residual carcass. (vii) Counting efficacy data shall be made available to the Agency upon request. [52 FR 19868, May 28, 1987] 795.250 Developmental neurotoxicity screen. (a) Purpose. In the assessment and evaluation of the toxic characteristics of a chemical, it is important to determine when acceptable exposures in the adult may not be acceptable to a developing organism. This test is designed to provide information on the potential functional and morphologic hazards to the nervous system which may arise in the offspring from exposure of the mother during pregnancy and lactation. (b) Principle of the test method. The test substance is administered to several groups of pregnant animals during gestation and lactation, one dose level being used per group. Offspring are randomly selected from within litters for neurotoxicity evaluation. The evaluation includes observation to detect gross neurological and behavioral abnormalities, determination of motor activity, neuropathological evaluation, and brain weights. Measurements are carried out periodically during both postnatal development and adulthood. (c) Test procedures--(1) Animal selection--(i) Species and strain. Testing should be performed in the Sprague Dawley rat. (ii) Age. Young adult animals (nulliparous females) shall be used. (iii) Sex. Pregnant females shall be used at each dose level. (iv) Number of animals. The objective is for a sufficient number of pregnant rats to be exposed to ensure that an adequate number of offspring are produced for neurotoxicity evaluation. At least 20 litters are recommended at each dose level. This number assumes a coefficient of variation of 20 to 25 percent for most behavioral tests. If, based upon experience with historical control data or data for positive controls in a given laboratory, the coefficient of variation for a given task is higher than 20 to 25 percent, then calculation of appropriate sample sizes to detect a 20 percent change from control values with 80 percent power would need to be done. For most designs, calculations can be made according to Dixon and Massey (1957) under paragraph (e)(5) of this section, Neter and Wasserman (1974) under paragraph (e)(10) of this section, Sokal and Rohlf (1969) under paragraph (e)(11) of this section, or Jensen (1972) under paragraph (e)(8) of this section. (A) On day 4 after birth, the size of each litter should be adjusted by eliminating extra pups by random selection to yield, as nearly as possible, 4 males and 4 females per litter. Whenever the number of male or female pups prevents having 4 of each sex per litter, partial adjustment (for example, 5 males and 3 females) is permitted. Adjustments are not appropriate for litters of less than 8 pups. Elimination of runts only is not appropriate. Individual pups should be identified uniquely after standardization of litters. A method that may be used can be found in Adams et al. (1985) under paragraph (e)(1) of this section. (B) After standardization of litters, males and females shall be randomly assigned to one of each of three behavioral tasks. Alternatively, more than one of the behavioral tasks may be conducted in the same animal. In the latter case, a minimum of 1 to 2 days should separate the tests when conducted at about the same age. (C) One male and one female shall be randomly selected from each litter for sacrifice at weaning as specified in paragraph (c)(8) of this section. (2) Control group. A concurrent control group shall be used. This group shall be a sham treated group, or, if a vehicle is used in administering the test substance, a vehicle control group. Animals in the control groups shall be handled in an identical manner to test group animals. The vehicle shall neither be developmentally toxic nor have effects on reproduction. (3) Dose levels and dose selection. (i) At least 3 dose levels plus a control (vehicle control, if a vehicle is used) shall be used. (ii) If the substance has been shown to be developmentally toxic either in a standard developmental toxicity study or a pilot study, the highest dose level shall be the maximum dose which will not induce in utero or neonatal deaths or malformations sufficient to preclude a meaningful evaluation of neurotoxicity. (iii) In the absence of standard developmental toxicity, unless limited by the physicochemical nature or biologicial properties of the substance, the highest dose level shall induce some overt maternal toxicity but shall not result in a reduction in weight gain exceeding 20 percent during gestation and lactation. (iv) The lowest dose should not produce any grossly observable evidence of either maternal or developmental neurotoxicity. (v) The intermediate dose(s) shall be equally spaced between the highest and lowest dose. (4) Dosing period. Day 0 in the test is the day on which a vaginal plug and/or sperm are observed. The dose period shall cover the period from day 6 of gestation through weaning (21 days postnatally). (5) Administration of test substance. The test substance or vehicle should be administered orally by intubation. The test substance shall be administered at the same time each day. The animals shall be weighed periodically and the dosage based on the most recent weight determination. (6) Observation of dams. (i) A gross examination of the dams shall be made at least once each day, before daily treatment. The animals shall be observed by trained technicians who are blind with respect to the animal's treatment, using standardized procedures to maximize inter-observer reliability. Where possible, it is advisable that the same observer be used to evaluate the animals in a given study. If this is not possible, some demonstration of inter-observer reliability is required. (ii) During the treatment and observation periods, cage-side observations shall include: (A) Any responses with respect to body position, activity level, coordination of movement, and gait. (B) Any unusual or bizarre behavior including, but not limited to headflicking, head searching, compulsive biting or licking, self-mutilation, circling, and walking backwards. (C) The presence of: (1) Convulsions. (2) Tremors. (3) Increased levels of lacrimation and/or red-colored tears. (4) Increased levels of salivation. (5) Piloerection. (6) Pupillary dilation or constriction. (7) Unusual respiration (shallow, labored, dyspneic, gasping, and retching) and/or mouth breathing. (8) Diarrhea. (9) Excessive or diminished urination. (10) Vocalization. (iii) Signs of toxicity shall be recorded as they are observed, including the time of onset, the degree and duration. (iv) Animals shall be weighed at least weekly. (v) The day of delivery of litters shall be recorded. (7) Study conduct--(i) Observation of offspring. (A) All offspring shall be examined cage-side daily for gross signs of mortality and morbidity. (B) All offspring shall be examined outside the cage for gross signs of toxicity whenever they are weighed or removed from their cages for behavioral testing. The offspring shall be observed by trained technicians, who are blind with respect to the animal's treatment using standardized procedures to maximize inter-observer reliability. Where possible, it is advisable that the same observer be used to evaluate the animals in a given study. If this is not possible, some demonstration of inter-observer reliability is required. At a minimum, the end points outlined in paragraph (c)(6)(ii) of this section shall be monitored as appropriate for the developmental stage being observed. (C) Any gross signs of toxicity in the offspring shall be recorded as they are observed, including the time of onset, the degree, and duration. (ii) Developmental landmarks. Live pups should be counted and litters weighed by weighing each individual pup at birth, or soon thereafter, and on days 4, 7, 13, 17, and 21, and biweekly thereafter. The age of the pups at the time of the appearance of the following developmental landmarks shall be determined: (A) Vaginal opening. General procedure for this determination may be found in Adams et al. (1985) under paragraph (e)(1) of this section. (B) Testes descent. General procedure for this determination may be found in Adams et al. (1985) under paragraph (e)(1) of this section. (iii) Motor activity. (A) Motor activity shall be monitored specifically on days 13, 17, 21, 45 (÷2 days), and 60 (÷2 days). Motor activity shall be monitored by an automated activity recording apparatus. The device used shall be capable of detecting both increases and decreases in activity, i.e., baseline activity as measured by the device shall not be so low as to preclude decreases nor so high as to preclude increases. Each device shall be tested by standard procedures to ensure, to the extent possible, reliability of operation across devices and testing of animals within dose groups shall be balanced across devices. (B) Each animal shall be tested individually. The test session shall be long enough to demonstrate habituation of motor activity in control animals, i.e., to approach asymptotic levels by the last 20 percent of the session. Animals' activity counts shall be collected in equal time periods of no greater than 10 minutes duration. All sessions shall have the same duration. Treatment groups shall be counter-balanced across test times. (C) Efforts shall be made to ensure that variations in the test conditions are minimal and are not systematically related to treatment. Among the variables which can affect motor activity are sound level, size, and shape of the test cage, temperature, relative humidity, lighting conditions, odors, use of home cage or novel test cage, and environmental distractions. (D) Additional information on the conduct of a motor activity study may be obtained in the TSCA motor activity guideline, in 798.6200 of this chapter. (iv) Auditory startle test. An auditory startle habituation test shall be performed on the offspring on days 22 and 60. Details on the conduct of this testing may be obtained in Adams et al. (1985) under paragraph (e)(1) of this section. In performing the auditory startle task, the mean response amplitude on each block of 10 trials (5 blocks of 10 trials per session on each day of testing) shall be made. While use of pre-pulse inhibition is not a requirement, it may be used at the discretion of the investigator. Details on the conduct of this testing may be obtained from Ison (1984) under paragraph (e)(7) of this section. (v) Active avoidance test. Active avoidance testing shall be conducted beginning at 60 to 61 days of age. Details on the apparatus may be obtained in Brush and Knaff (1959) and on the conduct of testing from Brush (1962), under paragraphs (e)(2) and (e)(4) of this section, respectively; reviews on active avoidance conditioning by Brush (1971) and McAllister and McAllister (1971) can be found under paragraphs (e)(3) and (e)(9) of this section, respectively. In performing the active avoidance task, the following measures should be made: (A) Mean number of shuttles during the adaptation period preceding each daily session. (B) Mean number and latency of avoidances per session, presented in blocks of 10 trials (2 blocks of 10 trials per session across 5 sessions). (C) Mean number and latency of escapes per session, presented in blocks of 10 trials as above. (D) Mean duration of shocks per session, presented in blocks of 10 trials as above. (E) Mean number of shuttles during the inter-trial intervals. (8) Post-mortem evaluation--(i) Age of animals. One male and one female per litter shall be sacrificed at weaning and the remainder following the last behavioral measures. Neuropathology and brain weight determinations shall be made on animals sacrificed at weaning and after the last behavioral measures. (ii) Neuropathology. Details for the conduct of neuropathology evaluation may be obtained in the TSCA neuropathology guideline, in 798.6400 of this chapter. At least 6 offspring per dose group shall be randomly selected from each sacrificed group (weaning and adulthood) for neuropathologic evaluation. These animals shall be balanced across litters, and equal numbers of males and females shall be used. The remaining sacrificed animals shall be used to determine brain weight. Animals shall be perfused in situ by a generally recognized technique. After perfusion, the brain and spinal cord shall be removed and gross abnormalities noted. Cross-sections of the following areas shall be examined: The forebrain, the center of the cerebrum and midbrain, the cerebellum and pons, and the medulla oblongata; the spinal cord at cervical and lumbar swelling; Gasserian ganglia, dorsal root ganglia, dorsal and ventral root fibers, proximal sciatic nerve (mid-thigh and sciatic notch), sural nerve (at knee), and tibial nerve (at knee). Tissue samples from both the central and peripheral nervous system shall be further immersion-fixed and stored in appropriate fixative for further examination. After dehydration, tissue specimens shall be cleared with xylene and embedded in paraffin or paraplast except for the sural nerve which should be embedded in plastic. A method for plastic embedding is described by Spencer et al. under paragraph (e)(12) of this section. Tissue sections shall be prepared from the tissue blocks. The following general testing sequence is recommended for gathering histopathological data: (A) General staining. A general staining procedure shall be performed on all tissue specimens in the highest treatment group. Hematoxylin and eosin (H&E) shall be used for this purpose. The staining shall be differentiated properly to achieve bluish nuclei with pinkish background. (B) Special stains. Based on the results of the general staining, selected sites and cellular components shall be further evaluated by use of specific techniques. If H&E screening does not provide such information, a battery of stains shall be used to assess the following components in all appropriate required samples: Neuronal body (e.g., Einarson's gallocyanin), axon (e.g., Kluver's Luxol Fast Blue), and neurofibrils (e.g., Bielchosky). In addition, nerve fiber teasing shall be used. A section of normal tissue shall be included in each staining to assure that adequate staining has occurred. Any changes shall be noted and representative photographs shall be taken. If lesions are observed, the special techniques shall be repeated in the next lower treatment group until no further lesions are detectable. (C) Alternative technique. If the anatomical locus of expected neuropathology is well-defined, epoxy-embedded sections stained with toluidine blue may be used for small sized tissue samples. This technique obviates the need for special stains. (iii) Brain weight. At least 10 animals that are not sacrificed for histopathology shall be used to determine brain weight. The animals shall be decapitated and the brains carefully removed, blotted, chilled, and weighed. The following dissection shall be performed on an ice-cooled glass plate: First, the rhombencephalon is separated by a transverse section from the rest of the brain and dissected into the cerebellum and the medulla oblongata/pons. A transverse section is made at the level of the ``optic chiasma'' which delimits the anterior part of the hypothalamus and passes through the anterior commissure. The cortex is peeled from the posterior section and added to the anterior section. This divides the brain into four sections, the telencephalon, the diencephalon/mid-brain, the medulla oblongata/pons, and the cerebellum. Sections shall be weighed as soon as possible after dissection to avoid drying. Detailed methodology is available in Glowinski and Iversen (1966) under paragraph (e)(6) of this section. (d) Data reporting and evaluation. In addition to the reporting requirements specified in part 792, subpart J of this chapter, the final test report shall include the following information. (1) Description of system and test methods. (i) A detailed description of the procedures used to standardize observation and operational definitions for scoring observations. (ii) Positive control data from the laboratory performing the test that demonstrate the sensitivity of the procedures being used. These data do not have to be from studies using prenatal exposures. However, the laboratory must demonstrate competence in testing neonatal animals perinatally exposed to chemicals and establish test norms for the appropriate age group. (iii) Procedures for calibrating and assuring the equivalence of devices and balancing treatment groups. (iv) A short justification explaining any decisions where professional judgement is involved such as fixation technique and choice of stains. (2) Results. The following information shall be arranged by test group dose level. (i) In tabular form, data for each animal shall be provided showing: (A) Its identification number and litter from which it came. (B) Its body weight and score on each developmental landmark at each observation time; total session activity counts and intrasession subtotals on each day measured; auditory startle response magnitude session counts and intrasession subtotals on each day measured; avoidance session counts and intrasession counts on each day measured; time and cause of death (if appropriate); locations, nature or frequency, and severity of the lesions; total brain weight; absolute weight of each of the four sections; and weight of each section as a percentage of total brain weight. A commonly used scale such as 1+, 2+, 3+, and 4+ for degree of severity of lesions ranging from very slight to extensive may be used for morphologic evaluation. Any diagnoses derived from neurologic signs and lesions, including naturally occurring diseases or conditions, shall also be recorded. (ii) Summary data for each group shall include: (A) The number of animals at the start of the test. (B) Body weights of the dams during gestation and lactation. (C) Litter size and mean weight at birth. (D) The number of animals showing each observation score at each observation time. (E) The percentage of animals showing each abnormal sign at each observation time. (F) The mean and standard deviation for each continuous end point at each observation time. These will include body weight, motor activity counts, acoustic startle responses, performance in active avoidance tests, and brain weights (both absolute and relative). (G) The number of animals in which any lesion was found. (H) The number of animals affected by each different type of lesion, the average grade of each type of lesion, and the frequency of each different type and/or location of lesions. (3) Evaluation of data. An evaluation of the test results shall be made. The evaluation shall include the relationship between the doses of the test substance and the presence or absence, incidence, and severity of any neurotoxic effect. The evaluation shall include appropriate statistical analyses. The choice of analyses shall consider tests appropriate to the experimental design and needed adjustments for multiple comparisons. (e) References. For additional background information on this test guideline, the following references should be consulted: (1) Adams, J., Buelke-Sam, J., Kimmel, C.A., Nelson, C.J., Reiter, L.W., Sobotka, T.J., Tilson, H.A., and Nelson, B.K. ``Collaborative behavioral teratology study: Protocol design and testing procedure.'' Neurobehavioral Toxicology and Teratology. 7: 579 - 586. (1985). (2) Brush, F.R. ``The effects of inter-trial interval on avoidance learning in the rat.'' Journal of Comparative Physiology and Psychology. 55: 888 - 892. (1962). (3) Brush, F.R. ``Retention of aversively motivated behavior.'' In: ``Adverse Conditioning and Learning.'' Brush, F.R., ed., New York: Academic Press. (1971). (4) Brush, F.R. and Knaff, P.R. ``A device for detecting and controlling automatic programming of avoidance-conditioning in a shuttle-box.'' American Journal of Psychology. 72: 275 - 278 (1959). (5) Dixon, W.J. and Massey, E.J. ``Introduction to Statistical Analysis.'' 2nd ed. New York: McGraw-Hill. (1957). (6) Glowinski, J. and Iversen, L.L. ``Regional studies of catecholamines in the rat brain-I.'' Journal of Neurochemistry. 13: 655 - 669. (1966). (7) Ison, J.R. ``Reflex modification as an objective test for sensory processing following toxicant exposure.'' Neurobehavioral Toxicology and Teratology. 6: 437 - 445. (1984). (8) Jensen, D.R. ``Some simultaneous multivariate procedures using Hotelling's T2 Statistics.'' Biometrics. 28: 39 - 53. (1972). (9) McAllister, W.R. and McAllister, D.E. ``Behavioral measurement of conditioned fear.'' In: ``Adverse Conditioning and Learning.'' Brush, F.R., ed., New York: Academic Press (1971). (10) Neter, J. and Wasserman, W. ``Applied Linear Statistical Models.'' Homewood: Richard D. Irwin, Inc. (1974). (11) Sokal, R.P. and Rohlf, E.J. ``Biometry.'' San Francisco: W.H. Freeman and Co. (1969). (12) Spencer, P.S., Bischoff, M.C., and Schaumburg, H.H., ``Neuropathological methods for the detection of neurotoxic disease.'' In: ``Experimental and Clinical Neurotoxicology.'' Spencer, P.S. and Schaumburg, H.H., eds., Baltimore, MD: Williams & Wilkins, pp. 743 - 757. (1980). [53 FR 5957, Feb. 26, 1988] 795.260 Subchronic oral toxicity test. (a) Purpose. In the assessment and evaluation of the toxic characteristics of a test substance, the determination of subchronic oral toxicity may be carried out after initial information on toxicity has been obtained by acute testing. The subchronic oral study has been designed to permit the determination of the no-observed-effect level and toxic effects associated with continuous or repeated exposure to a test substance for a period of 90 days. The test is not capable of determining those effects that have a long latency period for development (e.g., carcinogenicity and life shortening). It provides information on health hazards likely to arise from repeated exposure by the oral route over a limited period of time. It will provide information on target organs, the possibilities of accumulation, and can be of use in selecting dose levels for chronic studies and for establishing safety criteria for human exposure. (b) Definitions. (1) Subchronic oral toxicity is the adverse effects occurring as a result of the repeated daily exposure of experimental animals to a chemical for a part (approximately 10 percent for rats) of a life span. (2) Dose is the amount of test substance administered. Dose is expressed as weight of test substance (g, mg) per unit weight of test animal (e.g., mg/kg), or as weight of test substance per unit weight of food or drinking water. (3) No-effect level/No-toxic-effect level/No-adverse-effect level/No-observed-effect level is the maximum dose used in a test which produces no observed adverse effects. A no-observed-effect level is expressed in terms of the weight of a substance given daily per unit weight of test animal (mg/kg). When administered to animals in food or drinking water, the no-observed-effect level is expressed as mg/kg of food of mg/ml of water. (4) Cumulative toxicity is the adverse effects of repeated doses occurring as a result of prolonged action on, or increased concentration of, the administered substance or its metabolites in susceptible tissue. (c) Principle of the test method. The test substance is administered orally in graduated daily doses to several groups of experimental animals, one dose level per group, for a period of 90 days. During the period of administration the animals are observed daily to detect signs of toxicity. Animals which die during the period of administration are necropsied. At the conclusion of the test all animals are necropsied and histopathological examinations carried out. (d) Test procedures--(1) Animal selection-- (i) Species. Rats and mice shall be used. (ii) Age. (A) Young adult animals shall be employed. At the commencement of the study the weight variation of animals used shall not exceed ÷ 20 percent of the mean weight for each sex. (B) Dosing shall begin as soon as possible after weaning, ideally before the animals are 6 weeks old, and in any case not more than 8 weeks old. (iii) Sex. (A) Equal numbers of animals of each sex should be used at each dose level. (B) The females should be nulliparous and non-pregnant. (iv) Numbers. (A) At least 20 rats and 20 mice (10 females and 10 males of each species) shall be used at each dose level. (B) If interim sacrifices are required, the number shall be increased by the number of animals scheduled to be sacrificed before the completion of the study. (2) Control groups. A concurrent control group is required. This group shall be an untreated or sham-treated control group or, if a vehicle is used in administering the test substance, a vehicle control group. If the toxic properties of the vehicle are not known or cannot be made available, both untreated and vehicle control groups are required. (3) Satellite group. A satellite group of 20 rats and 20 mice (10 females and 10 males of each species) shall be treated with the high dose level for 90 days and observed for reversibility, persistence, or delayed occurrence of toxic effects for a post-treatment period of not less than 28 days. (4) Dose levels and dose selection. (i) In subchronic toxicity tests, it is desirable to have a dose response relationship as well as no-observed-toxic-effect level. Therefore, at least three dose levels with a control and, where appropriate, a vehicle control (corresponding to the concentration of vehicle at the highest exposure level) shall be used. Doses should be spaced appropriately to produce test groups with a range of toxic effects. The data shall be sufficient to produce a dose-response curve. (ii) The highest dose level shall result in toxic effects but not produce an incidence of fatalities which would prevent a meaningful evaluation. (iii) The lowest dose level shall not produce any evidence of toxicity. Where there is a usable estimation of human exposure the lowest dose level shall exceed this. (iv) Ideally, the intermediate dose level(s) should produce minimal observable toxic effects. If more than one intermediate dose is used, the dose levels should be spaced to produce a gradation of toxic effects. (v) The incidence of fatalities in low and intermediate dose groups and in the controls should be low to permit a meaningful evaluation of the results. (5) Exposure conditions. Ideally the animals should be dosed with the test substance on a 7-day per week basis over a period of 90 days. However, based primarily on practical considerations, dosing by gavage or capsule studies on a 5-day per week basis shall be acceptable. (6) Observation period. (i) Duration of observation shall be for at least 90 days. (ii) Animals in the satellite group scheduled for followup observations shall be kept for not less than 28 days without treatment to detect recovery from, or persistence of, toxic effects. (7) Administration of the test substance. (i) The test substance shall be administered in the diet or in capsules. Alternatively, it may be administered by gavage or in the drinking water. (ii) All animals shall be dosed by the same method during the entire experimental period. (iii) Where necessary, the test substance is dissolved or suspended in a suitable vehicle. If a vehicle or diluent is needed, ideally it should not elicit important toxic effects itself nor substantially alter the chemical or toxicological properties of the test substance. It is recommended that wherever possible the usage of an aqueous solution be considered first, followed by consideration of a solution of oil, and then by possible solution in other vehicles. (iv) For substances of low toxicity, it is important to ensure that when administered in the diet the quantities of the test substance involved do not interfere with normal nutrition. When the test substance is administered in the diet, either a constant dietary concentration (ppm) or a constant dose level in terms of the animals' body weight shall be used; the alternative used shall be specified. (v) For a substance administered by gavage or capsule, the dose shall be given at similar times each day, and adjusted at intervals (weekly or biweekly) to maintain a constant dose level in terms of animal body weight. (8) Observation of animals. (i) Each animal shall be handled and its physical condition appraised at least once each day. (ii) Additional observation shall be made daily with appropriate actions taken to minimize loss of animals to the study (e.g., necropsy or refrigeration of those animals found dead and isolation or sacrifice of weak or moribund animals). (iii) Signs of toxicity shall be recorded as they are observed including the time of onset, degree, and duration. (iv) Cage-side observations shall include, but not be limited to, changes in skin and fur, eyes and mucous membranes, respiratory, circulatory, autonomic and central nervous systems, somatomotor activity, and behavior pattern. (v) Measurements shall be made weekly of food consumption or water consumption when the test substance is administered in the food or drinking water, respectively. (vi) Animals shall be weighed weekly. (vii) At the end of the 90-day period all survivors in the nonsatellite treatment group shall be sacrificed. Moribund animals shall be removed and sacrificed when noticed. (9) Clinical examinations. (i) The following examinations shall be made on at least five animals of each sex in each group of rats. (A) Certain hematology determinations shall be carried out just prior to terminal sacrifice at the end of the test period. The following hematology determinations shall be carried out: hematocrit, hemoglobin concentration, erythrocyte count, total and differential leucocyte count, and a measure of clotting potential such as clotting time, prothrombin time, thromboplastin time, or platelet count. (B) Certain clinical biochemistry determinations shall be carried out just prior to terminal sacrifice at the end of the test period. The following clinical biochemical test areas shall be carried out: Electrolyte balance, carbohydrate metabolism, and liver and kidney function. The selection of additional tests shall be influenced by observations on the mode of action of the substance. Suggested additional determinations include: Calcium, phosphorus, chloride, sodium, potassium, fasting glucose (with period of fasting appropriate to the species/breed), serum glutamic-pyruvic transaminase (now known as serum alanine aminotransferase), serum glutamic oxaloacetic transaminase (now known as serum aspartate aminotransferase), ornithine decarboxylase, gamma glutamyl transpeptidase, urea nitrogen, albumen, blood creatinine, total bilirubin, and total serum protein measurements. Other determinations which may be necessary for an adequate toxicological evaluation include analyses of lipids, hormones, acid/base balance, methemoglobin, and cholinesterase activity. Additional clinical biochemistry may be employed where necessary to extend the investigation of observed effects. (ii) The following examinations shall be made on at least five animals of each sex in each group. (A) Ophthalmological examination, using an ophthalmoscope or equivalent suitable equipment, shall be made prior to the administration of the test substance and at the termination of the study. If changes in the eyes are detected, all animals shall be examined. (B) Urinalysis is required only when there is an indication based on expected or observed toxicity. (10) Gross necropsy. (i) All animals shall be subjected to a full gross necropsy which includes examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents. (ii) At least the liver, kidneys, adrenals, gonads, and brain shall be weighed wet, as soon as possible after dissection to avoid drying. (iii) The following organs and tissues, or representative samples thereof, shall be preserved in a suitable medium for possible future histopathological examination: All gross lesions; brain--including sections of medulla/pons, cerebellar cortex and cerebral cortex; pituitary; thyroid/parathyroid; thymus; lungs; trachea; heart; sternum with bone marrow; salivary glands; liver; spleen; kidneys/adrenals; pancreas; gonads; uterus; accessory genital organs (epididymis, prostrate, and, if present, seminal vesicles); aorta; (skin), (non-rodent gall bladder); esophagus; stomach; duodenum; jejunum; ileum; cecum; colon; rectum; urinary bladder; representative lymph node; (mammary gland), (thigh musculature), peripheral nerve; (eyes), (femur including articular surface), (spinal cord at three levels--cervical, midthoracic and lumbar); and, (rodent-exorbital lachrymal glands). (11) Histopathology. (i) Full histopathology shall be performed on the organs and tissues, listed under paragraphs (d)(10) (ii) and (iii) of this section of all animals in the control and high-dose groups, and all animals that died or were killed during the study. (ii) Histopathology shall be performed on all gross lesions in all animals. (iii) Histopathology shall be performed on target organs in all animals. (iv) Histopathology shall be performed on the tissues mentioned in brackets under paragraph (d)(10)(iii) of this section if indicated by signs of toxicity or target organ involvement. (v) Histopathology shall be performed on lungs, liver, and kidneys of all animals. Special attention to examination of the lungs should be made for evidence of infection since this provides a convenient assessment of the state of health of the animals. (vi) For the satellite group, histopathology shall be performed on tissues and organs identified as showing effects in the treated groups. (e) Data and reporting--(1) Treatment of results. (i) Data shall be summarized in tabular form, showing for each test group the number of animals at the start of the test, the number of animals showing lesions, the type of lesions, and the percentage of animals displaying each type of lesion. (ii) All observed results, quantitative and incidental, shall be evaluated by an appropriate statistical method. Any generally acceptable statistical methods may be used; the statistical methods should be selected during the design of the study. (2) Evaluation of the study results. (i) The findings of a subchronic oral toxicity study should be evaluated in conjunction with the findings of preceding studies and considered in terms of the toxic effects and the necropsy and histopathological findings. The evaluation shall include the relationship between the dose of the test substance and the presence or absence, the incidence and severity, of abnormalities, including behavioral and clinical abnormalities, gross lesions, identified target organs, body weight changes, effects on mortality and any other general or specific toxic effects. The test shall provide a satisfactory estimation of a no-effect level. (ii) In any study which demonstrates an absence of toxic effects, further investigation to establish absorption and bioavailability of the test substance shall be considered. (3) Test report. In addition to the reporting requirements as specified in the TSCA Good Laboratory Practice Standards, subpart J of part 792 of this chapter, the following specific information shall be reported: (i) Group animal data. Tabulation of toxic response data by species, strain, sex, and exposure level for: (A) Number of animals dying. (B) Number of animals showing signs of toxicity. (C) Number of animals exposed. (ii) Individual animal data. (A) Time of death during the study or whether animals survived to termination. (B) Time of observation of each abnormal sign and its subsequent course. (C) Body weight data. (D) Food consumption data when collected. (E) Hematological tests employed and all results. (F) Clinical biochemistry tests employed and all results. (G) Necropsy findings. (H) Detailed description of all histopathological findings. (I) Statistical treatment of results where appropriate. [51 FR 40328, Nov. 6, 1986] 795.285 Morphologic transformation of cells in culture. (a) Purpose. In vitro assays for cellular transformation are semi-quantitative assays for the ability of chemical agents to morphologically alter (transform) cells in culture. Such transformation is associated with certain phenotypic changes such as loss of contact inhibition and the ability to form colonies in soft agar medium. The process by which these changes occur is assumed to be closely related to the process of in vivo carcinogenesis. Morphologically transformed cells appear as foci of dense, piled-up, altered cells on an underlying monolayer of normal cells. Three types of foci have been recognized. Type III foci appear to be most closely correlated with in vivo tumor formation. The ultimate criterion for morphologic transformation is the ability of the transformed cells to induce tumors when inoculated into appropriate hosts. Not all cells which appear to be morphologically transformed are capable of tumor formation. In general, there is reasonably good correlation between in vitro transformation and in vivo oncogenesis, although the correlation varies depending on the system being studied. These systems are believed to be reasonably good predictors of in vivo activity, and positive results are viewed as potential indications of in vivo carcinogenesis. (b) Definitions. (1) Morphologic transformation is the acquisition of certain phenotypic characteristics, most notably loss of contact inhibition and loss of anchorage dependence, which are often but not always associated with the ability to induce tumors in appropriate hosts. (2) Type III foci of transformed cells are multilayered aggregations of densely staining cells with random orientation and criss-cross arrays at the periphery of the aggregate. They appear as dark stained areas on a light staining background monolayer which is one-cell thick. (c) Reference substances. Not applicable. (d) Test method--(1) Principle. (i) Three systems for detecting chemically induced morphologic transformation have been described. They are: (A) Systems which employ cell lines (cells with an indefinite lifespan). (B) Systems which employ cell strains (cells with a finite or limited lifespan). (C) Systems which detect the interaction between chemicals and oncogenic viruses. (ii) This study shall employ an established cell line for detection of morphologic transformaton. (2) Description. Cells in culture are exposed to the test substance, both with and without metabolic activation, for a defined period of time. Cytotoxicity is determined by measuring the colony-forming ability and growth rate of the cultures after the treatment period. At the end of the treatment period, cultures are maintained in growth medium for a sufficient period of time to allow near-optimal expression of transformed foci. (3) Cells. (i) Balb/c - 3T3 mouse cells originally obtained from clone A - 31 or its derivatives shall be used in the assay. Cells shall be checked for mycoplasm contamination prior to use in the assay and may be checked for karyotype. (ii) Appropriate culture media and incubation conditions (culture vessels, CO2 concentrations, temperature, and humidity) shall be used. (4) Metabolic activation. Cells shall be exposed to test substance both in the presence and absence of a metabolic activation system. The metabolic activation system shall be derived from primary cultures of rat hepatocytes. (5) Control groups. Positive and negative (untreated and vehicle) controls shall be included in each experiment. 3-Methylcholanthrene is an example of a positive control for experiments without metabolic activation. Dimethylnitrosamine is an example of a positive control in experiments with metabolic activation. (6) Test chemicals--(i) Vehicle. Test agents shall be dissolved in serum-complete culture medium prior to treatment of the cells. (ii) Exposure concentrations. Several concentrations (usually at least four) of the test substance shall be used. These shall be selected on the basis of a preliminary cytotoxicity assay performed both with and without metabolic activation. The highest concentration shall produce a low level of survival (approximately 10 to 20 percent), and the survival in the lowest concentration shall approximate that of the negative control. (e) Test performance. (1) Cells shall be exposed to the test substance both with and without metabolic activation. Exposure shall be for 72 hours for experiments without metabolic activation and for 48 hours for experiments with metabolic activation unless different exposure times are justified by the investigator. (2) At the end of the exposure period, cells shall be washed and cultured to determine viability and to allow for expression of transformation. (3) At the end of the incubation period (generally 4 to 6 weeks), cells shall be fixed and stained, and the number of transformed (Type III) foci shall be enumerated. (4) All results shall be confirmed in an independent experiment if a single, statistically significant positive effect is produced at one dose point without a dose response. A positive response should be confirmed by testing over a narrow range of concentrations. (5) Tumorigenic potential of isolated morphologically transformed foci may be determined by inoculation into suitable hosts. (f) Data and report--(1) Treatment of results. (i) Data shall be presented in tabular form. Individual colony counts for the treated and control groups shall be presented for both transformation and survival. (ii) Survival and cloning efficiencies shall be given as a percentage of the controls. Transformation shall be expressed as a number of foci per dish, the number of dishes with transformed foci, and the number of transformed foci per number of surviving cells. (2) Interpretation of results. (i) There are several criteria for determining a positive result, one of which is a statistically significant concentration-related increase in the number of transformed foci. Another criterion may be based upon the detection of a reproducible and statistically significant positive response for at least one of the test substance concentrations. (ii) A test substance which does not produce either a statistically significant concentration-related increase in the number of transformed foci or a statistically significant and reproducible positive response at any one of the test points is considered to be negative in this system. (iii) Both biological and statistical significance should be considered together in the evaluation. (3) Test evaluation. (i) Positive results for an in vitro mammalian cell transformation assay indicate that, under the test conditions, the test substance induces morphologic transformation in the cultured mammalian cells used. (ii) Negative results indicate that, under the test conditions, the test substance does not induce morphologic transformation in the cultured mammalian cells used. (4) Test report. In addition to the reporting recommendations as specified under 40 CFR part 792 subpart J, the following specific information shall be reported: (i) Cell type used, including subclone designation and passage number; number of cell cultures; methods used for maintenance of cell cultures. (ii) Rationale for selection of concentrations and number of cultures. (iii) Test conditions: Composition of media, CO2 concentration, concentration of test substance, vehicle, incubation temperature, incubation time, duration of treatment, cell density during treatment, type of metabolic activation system, positive and negative controls, length of expression period (including number of cells seeded and subculture and feeding schedules, if appropriate). (iv) Methods used to enumerate numbers of viable cells and transformed foci. (v) Dose-response relationship, where possible. (g) References. For additional background information on this test guideline, the following references should be consulted: (1) Heidelberger, C., Freeman, A.E., Pienta, R.J., Sivak, A., Bertram, J.S., Castro, B.C., Dunkel, V.C., Francis, M.W., Kakunaga, T., Little, J.B., Schechtman, L.M., ``Cell transformation by chemical agents--a review and analysis of the literature: a report of the U.S. Environmental Protection Agency Gene-Tox Program.'' Mutation Research 114:283 - 385, 1983. (2) Kakunaga, T. ``A quantitative system for assay of malignant transformation by carcinogens using a clone derived from Balb-3T3.'' International Journal of Cancer 12:463 - 473, 1973. (3) Reznikoff, C.A., Bertram, J.S., Brankow, D.W., Heidelberger, C. ``Quantitative and qualitative studies of chemical transformation of cloned C3H mouse embryo cells sensitive to postconfluence inhibition of cell division.'' Cancer Research 33:3239 - 3249, 1973. (4) Reznikoff, C.A., Brankow, D.W., Heidelberger, C. ``Establishment and characterization of a cloned line of C3H mouse embryo cells sensitive to postconfluence inhibition of division.'' Cancer Research 33:3231 - 3238, 1973. (5) Sivak, A., Charest, M.C., Dudenko, L., Silveira, D.M., Simons, I., Wild, A.W. ``Balb/c - 3T3 cells as target cells for chemically induced neoplastic transformation.'' In: Advances in modern environmental toxicology, mammalian cell transformation by chemical carcinogens, Vol. I. Mishra, N., Dunkel, V., Mehlman, M., eds. Princeton Junction, NJ: Senate Press, pp. 133 - 180, 1981. (6) Sivak, A., Tu, A.S. ``Factors influencing neoplastic transformation by chemical carcinogens in Balb/c-3T3 cells.'' In: The predictive value of short-term screening tests in carcinogenicity evaluation. Williams, G.M., Kroes, R., Waaijers, H.W., Van de Poll, K.W., eds. Amsterdam, New York, Oxford: Elsevier/North Holland Biomedical Press, pp. 177 - 190, 1980. (7) Williams, G.M. ``Detection of chemical carcinogens by unscheduled DNA synthesis in rat liver primary cell culture.'' Cancer Research 37:1845 - 1851, 1977. [52 FR 19085, May 20, 1987]