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Electronic Code of Federal Regulations

e-CFR data is current as of December 12, 2019

Title 40Chapter ISubchapter RPart 795 → Subpart D


Title 40: Protection of Environment
PART 795—PROVISIONAL TEST GUIDELINES


Subpart D—Provisional Health Effects Guidelines


Contents
§795.225   Dermal pharmacokinetics of DGBE and DGBA.
§795.228   Oral/dermal pharmacokinetics.
§795.231   Pharmacokinetics of isopropanal.
§795.232   Inhalation and dermal pharmacokinetics of commercial hexane.
§795.250   Developmental neurotoxicity screen.

§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 cm2 for rats, 5 cm2 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]

§795.231   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 110 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 he 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 1863-83 is available for public inspection at the National Archives and Records Administration (NARA). For information on the availability of this material at NARA, call 202-741-6030, or go to: http://www.archives.gov/federal__register/code__of__federal__regulations/ibr__locations.html.\n. Copies are available at the addresses in §700.17(b)(1) and (2) of this chapter. 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.

[55 FR 632, Jan. 8, 1990, as amended at 58 FR 34205, June 23, 1993; 60 FR 34466, July 3, 1995; 69 FR 18803, Apr. 9, 2004; 77 FR 46293, Aug. 3, 2012]

§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]

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