3 1 0 He a lt h 2 Fire 3 Re a c t iv it y 0 P e rs o n a l P ro t e c t io n G Material Safety Data Sheet Hexanes MSDS Section 1: Chemical Product and Company Identification Product Name: Hexanes Contact Information: Sciencelab.com, Inc. 14025 Smith Rd. Houston, Texas 77396 Catalog Codes: SLH2335, SLH2032 CAS#: 110-54-3 US Sales: 1-800-901-7247 International Sales: 1-281-441-4400 RTECS: MN9275000 TSCA: TSCA 8(b) inventory: Hexane Order Online: ScienceLab.com CI#: Not applicable. Synonym: CHEMTREC (24HR Emergency Telephone), call: 1-800-424-9300 Chemical Name: Hexane International CHEMTREC, call: 1-703-527-3887 Chemical Formula: C6-H14 For non-emergency assistance, call: 1-281-441-4400 Section 2: Composition and Information on Ingredients Composition: Name CAS # % by Weight Hexanes 110-54-3 98.5-99.9 Toxicological Data on Ingredients: Hexane: ORAL (LD50): Acute: 25000 mg/kg [Rat]. Section 3: Hazards Identification Potential Acute Health Effects: Hazardous in case of skin contact (permeator), of ingestion, of inhalation. Slightly hazardous in case of skin contact (irritant), of eye contact (irritant). Potential Chronic Health Effects: CARCINOGENIC EFFECTS: Not available. MUTAGENIC EFFECTS: Mutagenic for bacteria and/or yeast. TERATOGENIC EFFECTS: Not available. DEVELOPMENTAL TOXICITY: Not available. The substance may be toxic to peripheral nervous system, skin, central nervous system (CNS). Repeated or prolonged exposure to the substance can produce target organs damage. Section 4: First Aid Measures Eye Contact: p. 1 Check for and remove any contact lenses. Immediately flush eyes with running water for at least 15 minutes, keeping eyelids open. Get medical attention if irritation occurs. Skin Contact: Wash with soap and water. Cover the irritated skin with an emollient. Get medical attention if irritation develops. Serious Skin Contact: Wash with a disinfectant soap and cover the contaminated skin with an anti-bacterial cream. Seek medical attention. Inhalation: If inhaled, remove to fresh air. If not breathing, give artificial respiration. If breathing is difficult, give oxygen. Get medical attention if symptoms appear. Serious Inhalation: Evacuate the victim to a safe area as soon as possible. Loosen tight clothing such as a collar, tie, belt or waistband. If breathing is difficult, administer oxygen. If the victim is not breathing, perform mouth-to-mouth resuscitation. Seek medical attention. Ingestion: Do NOT induce vomiting unless directed to do so by medical personnel. Never give anything by mouth to an unconscious person. Loosen tight clothing such as a collar, tie, belt or waistband. Get medical attention if symptoms appear. Serious Ingestion: Not available. Section 5: Fire and Explosion Data Flammability of the Product: Flammable. Auto-Ignition Temperature: 225°C (437°F) Flash Points: CLOSED CUP: -22.5°C (-8.5°F). (TAG) Flammable Limits: LOWER: 1.15% UPPER: 7.5% Products of Combustion: These products are carbon oxides (CO, CO2). Fire Hazards in Presence of Various Substances: Highly flammable in presence of open flames and sparks, of heat. Non-flammable in presence of shocks. Explosion Hazards in Presence of Various Substances: Risks of explosion of the product in presence of mechanical impact: Not available. Risks of explosion of the product in presence of static discharge: Not available. Fire Fighting Media and Instructions: Flammable liquid, insoluble in water. SMALL FIRE: Use DRY chemical powder. LARGE FIRE: Use water spray or fog. Special Remarks on Fire Hazards: Extremely flammable liquid and vapor. Vapor may cause flash fire. Special Remarks on Explosion Hazards: Not available. Section 6: Accidental Release Measures Small Spill: Absorb with an inert material and put the spilled material in an appropriate waste disposal. Large Spill: Flammable liquid, insoluble in water. Keep away from heat. Keep away from sources of ignition. Stop leak if without risk. Absorb with DRY earth, sand or other non-combustible material. Do not get water inside container. Do not touch spilled material. Prevent entry into sewers, basements or confined areas; dike if needed. Call for assistance on disposal. Be careful that the product is not present at a concentration level above TLV. Check TLV on the MSDS and with local authorities. Section 7: Handling and Storage p. 2 Precautions: Keep locked up.. Keep away from heat. Keep away from sources of ignition. Ground all equipment containing material. Do not ingest. Do not breathe gas/fumes/ vapor/spray. Avoid contact with skin. Wear suitable protective clothing. In case of insufficient ventilation, wear suitable respiratory equipment. If ingested, seek medical advice immediately and show the container or the label. Keep away from incompatibles such as oxidizing agents. Storage: Store in a segregated and approved area. Keep container in a cool, well-ventilated area. Keep container tightly closed and sealed until ready for use. Avoid all possible sources of ignition (spark or flame). Section 8: Exposure Controls/Personal Protection Engineering Controls: Provide exhaust ventilation or other engineering controls to keep the airborne concentrations of vapors below their respective threshold limit value. Ensure that eyewash stations and safety showers are proximal to the work-station location. Personal Protection: Safety glasses. Lab coat. Vapor respirator. Be sure to use an approved/certified respirator or equivalent. Gloves (impervious). Personal Protection in Case of a Large Spill: Splash goggles. Full suit. Vapor respirator. Boots. Gloves. A self contained breathing apparatus should be used to avoid inhalation of the product. Suggested protective clothing might not be sufficient; consult a specialist BEFORE handling this product. Exposure Limits: TWA: 500 (ppm) from OSHA (PEL) [United States] Inhalation TWA: 1800 (mg/m3) from OSHA (PEL) [United States] Inhalation TWA: 176 (mg/m3) from ACGIH (TLV) [United States] SKIN TWA: 50 (ppm) from ACGIH (TLV) [United States] SKIN TWA: 500 STEL: 1000 (ppm) from ACGIH (TLV) [United States] Inhalation TWA: 1760 STEL: 3500 (mg/m3) from ACGIH (TLV) [United States] Inhalation Consult local authorities for acceptable exposure limits. Section 9: Physical and Chemical Properties Physical state and appearance: Liquid. Odor: Gasoline-like or petroleum-like (Slight.) Taste: Not available. Molecular Weight: 86.18g/mole Color: Clear Colorless. pH (1% soln/water): Not applicable. Boiling Point: 68°C (154.4°F) Melting Point: -95°C (-139°F) Critical Temperature: Not available. Specific Gravity: 0.66 (Water = 1) Vapor Pressure: 17.3 kPa (@ 20°C) Vapor Density: 2.97 (Air = 1) Volatility: Not available. Odor Threshold: 130 ppm Water/Oil Dist. Coeff.: The product is more soluble in oil; log(oil/water) = 3.9 Ionicity (in Water): Not available. p. 3 Dispersion Properties: See solubility in water, diethyl ether, acetone. Solubility: Soluble in diethyl ether, acetone. Insoluble in cold water, hot water. Section 10: Stability and Reactivity Data Stability: The product is stable. Instability Temperature: Not available. Conditions of Instability: Heat, ingnition sources, incompatibles. Incompatibility with various substances: Reactive with oxidizing agents. Corrosivity: Not available. Special Remarks on Reactivity: Hexane can react vigorously with strong oxidizers (e.g. chlorine, bromine, fluorine) Special Remarks on Corrosivity: Not available. Polymerization: Will not occur. Section 11: Toxicological Information Routes of Entry: Absorbed through skin. Dermal contact. Inhalation. Ingestion. Toxicity to Animals: WARNING: THE LC50 VALUES HEREUNDER ARE ESTIMATED ON THE BASIS OF A 4-HOUR EXPOSURE. Acute oral toxicity (LD50): 25000 mg/kg [Rat]. Acute toxicity of the gas (LC50): 48000 ppm 4 hours [Rat]. Chronic Effects on Humans: MUTAGENIC EFFECTS: Mutagenic for bacteria and/or yeast. May cause damage to the following organs: peripheral nervous system, skin, central nervous system (CNS). Other Toxic Effects on Humans: Very hazardous in case of ingestion, of inhalation. Hazardous in case of skin contact (permeator). Slightly hazardous in case of skin contact (irritant). Special Remarks on Toxicity to Animals: Not available. Special Remarks on Chronic Effects on Humans: May cause adverse reproductive effects based on animal data. May be tumorigenic based on animal data. May affect genetic material. Passes through the placental barrier in animal. Special Remarks on other Toxic Effects on Humans: Acute Potential Health Effects: Skin: May cause mild skin irritation. It can be absorbed through the skin in harmful amounts. Eyes: May cause mild eye irritation. Inhalation: May be harmful if inhaled. Inhalation of vapors may cause respiratory tract irritation. Overexposure may affect, brain, spinal cord, behavior/central and peripheral nervous systems (lightheadness, dizziness, hallucinations, paralysis, blurred vision, memory loss, headache, euphoria, general anesthetic, muscle weakness, numbness of the extremeties, asphyxia, unconciousness and possible death), metabolism, respiration, blood, cardiovascular system, gastrointestinal system (nausea) Ingestion: May be harmful if swallowed. May cause gastrointestinal tract irritation with abdominal pain and nausea. May also affect the liver, blood, brain, peripheral and central nervous systems. Symptoms of over exposure by ingestion are similar to that of overexposure by inhalation. Section 12: Ecological Information Ecotoxicity: Not available. BOD5 and COD: Not available. p. 4 Products of Biodegradation: Possibly hazardous short term degradation products are not likely. However, long term degradation products may arise. Toxicity of the Products of Biodegradation: The product itself and its products of degradation are not toxic. Special Remarks on the Products of Biodegradation: Not available. Section 13: Disposal Considerations Waste Disposal: Waste must be disposed of in accordance with federal, state and local environmental control regulations. Section 14: Transport Information DOT Classification: CLASS 3: Flammable liquid. Identification: : Hexane UNNA: 1208 PG: II Special Provisions for Transport: Not available. Section 15: Other Regulatory Information Federal and State Regulations: Connecticut hazardous material survey.: Hexanes Illinois toxic substances disclosure to employee act: Hexanes Illinois chemical safety act: Hexanes New York release reporting list: Hexanes Rhode Island RTK hazardous substances: Hexanes Pennsylvania RTK: Hexanes Florida: Hexanes Minnesota: Hexanes Massachusetts RTK: Hexanes Massachusetts spill list: Hexanes New Jersey: Hexanes New Jersey spill list: Hexanes Louisiana spill reporting: Hexanes TSCA 8(b) inventory: Hexanes SARA 313 toxic chemical notification and release reporting: Hexanes CERCLA: Hazardous substances.: Hexanes: 5000 lbs. (2268 kg) Other Regulations: OSHA: Hazardous by definition of Hazard Communication Standard (29 CFR 1910.1200). EINECS: This product is on the European Inventory of Existing Commercial Chemical Substances. Other Classifications: WHMIS (Canada): CLASS B-2: Flammable liquid with a flash point lower than 37.8°C (100°F). CLASS D-2B: Material causing other toxic effects (TOXIC). DSCL (EEC): R11- Highly flammable. R20- Harmful by inhalation. R38- Irritating to skin. R51/53- Toxic to aquatic organisms, may cause long-term adverse effects in the aquatic environment. R62- Possible risk of impaired fertility. R65- Harmful: may cause lung damage if swallowed. R67- Vapors may cause drowsiness or dizziness. S9- Keep container in a well-ventilated place. S16Keep away from sources of ignition - No smoking. S29- Do not empty into drains. S33- Take precautionary measures against static discharges. S36/37- Wear suitable protective clothing and gloves. S61- Avoid release to the environment. Refer to special instructions/Safety data sheets. S62- If swallowed, do not induce vomiting: seek medical advice immediately and show this HMIS (U.S.A.): Health Hazard: 2 Fire Hazard: 3 Reactivity: 0 Personal Protection: g National Fire Protection Association (U.S.A.): p. 5 Health: 1 Flammability: 3 Reactivity: 0 Specific hazard: Protective Equipment: Gloves (impervious). Lab coat. Vapor respirator. Be sure to use an approved/certified respirator or equivalent. Wear appropriate respirator when ventilation is inadequate. Safety glasses. Section 16: Other Information References: Not available. Other Special Considerations: Not available. Created: 10/10/2005 08:19 PM Last Updated: 05/21/2013 12:00 PM The information above is believed to be accurate and represents the best information currently available to us. However, we make no warranty of merchantability or any other warranty, express or implied, with respect to such information, and we assume no liability resulting from its use. Users should make their own investigations to determine the suitability of the information for their particular purposes. In no event shall ScienceLab.com be liable for any claims, losses, or damages of any third party or for lost profits or any special, indirect, incidental, consequential or exemplary damages, howsoever arising, even if ScienceLab.com has been advised of the possibility of such damages. p. 6 g National Toxicology Program Toxicity Report Series Number 86 NTP Technical Report on the Toxicity Studies of Cedarwood Oil (Virginia) (CAS No. 8000-27-9) Administered Dermally to F344/N Rats and B6C3F1/N Mice ember 2016 National Institutes of Health Public Health Service U.S. Department of Health and Human Services FOREWORD The National Toxicology Program (NTP) is an interagency program within the Public Health Service (PHS) of the Department of Health and Human Services (HHS) and is headquartered at the National Institute of Environmental Health Sciences of the National Institutes of Health (NIEHS/NIH). Three agencies contribute resources to the program: NIEHS/NIH, the National Institute for Occupational Safety and Health of the Centers for Disease Control and Prevention (NIOSH/CDC), and the National Center for Toxicological Research of the Food and Drug Administration (NCTR/FDA). Established in 1978, the NTP is charged with coordinating toxicological testing activities, strengthening the science base in toxicology, developing and validating improved testing methods, and providing information about potentially toxic substances to health regulatory and research agencies, scientific and medical communities, and the public. The Toxicity Study Report series began in 1991. The studies described in the Toxicity Study Report series are designed and conducted to characterize and evaluate the toxicologic potential of selected substances in laboratory animals (usually two species, rats and mice). Substances selected for NTP toxicity studies are chosen primarily on the basis of human exposure, level of production, and chemical structure. The interpretive conclusions presented in the Toxicity Study Reports are based only on the results of these NTP studies. Extrapolation of these results to other species, including characterization of hazards and risks to humans, requires analyses beyond the intent of these reports. Selection per se is not an indicator of a substance’s toxic potential. The NTP conducts its studies in compliance with its laboratory health and safety guidelines and FDA Good Laboratory Practice Regulations and must meet or exceed all applicable federal, state, and local health and safety regulations. Animal care and use are in accordance with the Public Health Service Policy on Humane Care and Use of Animals. Studies are subjected to retrospective quality assurance audits before being presented for public review. NTP Toxicity Study Reports are indexed in the NIH/NLM PubMed database and are available free of charge electronically on the NTP website (http://ntp.niehs.nih.gov). National Toxicology Program Toxicity Report Series Number 86 NTP Technical Report on the Toxicity Studies of Cedarwood Oil (Virginia) (CAS NO. 8000-27-9) Administered Dermally to F344/N Rats and B6C3F1/N Mice Natasha R. Catlin, Ph.D., Study Scientist National Toxicology Program Post Office Box 12233 Research Triangle Park, NC 27709 ember 2016 National Institutes of Health Public Health Service U.S. Department of Health and Human Services 2 CONTRIBUTORS National Toxicology Program Dynamac Corporation Evaluated and interpreted results and reported findings Prepared quality assessment audits N.R. Catlin, Ph.D., Study Scientist R.A. Herbert, D.V.M., Ph.D., Study Pathologist C.R. Blystone, Ph.D. P.M. Foster, Ph.D. M.J. Hooth, Ph.D. A.P. King-Herbert, D.V.M. G.E. Kissling, Ph.D. B.S. McIntyre, Ph.D. D.E. Malarkey, D.V.M., Ph.D. S.L. Smith-Roe, Ph.D. M.D. Stout, Ph.D. I.O. Surh, Ph.D. G.S. Travlos, D.V.M. M.K. Vallant, B.S., M.T. S. Waidyanatha, Ph.D. N.J. Walker, Ph.D. K.L. Witt, M.S. S. Brecher, Ph.D., Principal Investigator S. Iyer, B.S. V.S. Tharakan, D.V.M. Battelle Columbus Operations N. Wakamatsu, D.V.M., Ph.D. Conducted studies and evaluated pathology findings M.R. Hejtmancik, Ph.D., Principal Investigator C.A. Colleton, D.V.M. L.M. Fomby, D.V.M., Ph.D. M.J. Ryan, D.V.M., Ph.D. J.D. Toft, II, D.V.M., M.S. NTP Pathology Working Group Evaluated slides and contributed to pathology report (April 5, 2007) G.D. Hill, D.V.M., Ph.D., Coordinator ILS, Inc. S.A. Elmore, D.V.M., M.S. National Toxicology Program G.P. Flake, M.D. National Toxicology Program R.A. Herbert, D.V.M., Ph.D. National Toxicology Program D.E. Malarkey, D.V.M., Ph.D. National Toxicology Program J.B. Nold, D.V.M., Ph.D. GlaxoSmithKline National Toxicology Program NTP Pathology Peer Review (Special Report) Evaluated slides and contributed to pathology report (November 7, 2013) R.R. Moore, D.V.M., Ph.D., Coordinator Experimental Pathology Laboratories, Inc. Provided pathology review M.H. Hamlin, II, D.V.M., Principal Investigator M.M. Gruebbel, D.V.M., Ph.D. R.A. Miller, D.V.M., Ph.D. R.R. Moore, D.V.M., Ph.D. Experimental Pathology Laboratories, Inc. M.F. Cesta, D.V.M., Ph.D. National Toxicology Program G.P. Flake, M.D. National Toxicology Program M.M. Gruebbel, D.V.M., Ph.D. Experimental Pathology Laboratories, Inc. R.A. Herbert, D.V.M., Ph.D. National Toxicology Program K. Janardhan, B.V.Sc., M.V.Sc, Ph.D. ILS, Inc. Bridge Global Pharmaceutical Services, Inc. D.E. Malarkey, D.V.M., Ph.D. Provided SMVCE analysis R.A. Miller, D.V.M., Ph.D. B.J.T. Muir, Ph.D., Principal Investigator B. Atkinson, M.Sc. Y. Wang, M.S. National Toxicology Program Experimental Pathology Laboratories, Inc. Cedarwood Oil, NTP TOX 86 3 Social & Scientific Systems, Inc. Biotechnical Services, Inc. Provided statistical analyses Prepared Toxicity Study Report M.V. Smith, Ph.D., Principal Investigator L.J. Betz, M.S. S.F. Harris, B.S. S.R. Gunnels, M.A., Principal Investigator L.M. Harper, B.S. J.I. Irving, M.A.P. T.S. Kumpe, M.A. D.C. Serbus, Ph.D. 4 PEER REVIEW The draft report on the toxicity studies of cedarwood oil was evaluated by the reviewers listed below. These reviewers serve as independent scientists, not as representatives of any institution, company, or governmental agency. In this capacity, reviewers determine if the design and conditions of these NTP studies are appropriate and ensure that this Toxicity Study Report presents the experimental results and conclusions fully and clearly. Gregory L. Erexson, Ph.D. Preclinical Safety Research & Development AbbVie, Inc. North Chicago, IL Laura A. Hansen, Ph.D. Department of Biomedical Sciences Creighton University School of Medicine Omaha, NE 5 CONTENTS ABSTRACT ................................................................................................................................................................. 7 INTRODUCTION ..................................................................................................................................................... 13 Chemical and Physical Properties ........................................................................................................................... 13 Production, Use, and Human Exposure .................................................................................................................. 14 Regulatory Status .................................................................................................................................................... 15 Absorption, Distribution, Metabolism, and Excretion ............................................................................................ 15 Toxicity ................................................................................................................................................................... 15 Reproductive and Developmental Toxicity............................................................................................................. 16 Carcinogenicity ....................................................................................................................................................... 16 Genetic Toxicity...................................................................................................................................................... 16 Study Rationale ....................................................................................................................................................... 16 MATERIALS AND METHODS .............................................................................................................................. 17 Procurement and Characterization .......................................................................................................................... 17 Preparation and Analysis of Dose Formulations ..................................................................................................... 18 Animal Source ........................................................................................................................................................ 19 Animal Welfare ....................................................................................................................................................... 19 3-Month Studies ...................................................................................................................................................... 19 Statistical Methods .................................................................................................................................................. 23 Quality Assurance Methods .................................................................................................................................... 24 Genetic Toxicology ................................................................................................................................................. 24 RESULTS ................................................................................................................................................................... 27 3-Month Study in Rats ............................................................................................................................................ 27 3-Month Study in Mice ........................................................................................................................................... 35 Genetic Toxicology ................................................................................................................................................. 42 DISCUSSION ............................................................................................................................................................. 43 REFERENCES .......................................................................................................................................................... 47 APPENDIXES Appendix A Summary of Nonneoplastic Lesions in Rats and Mice ............................................................ A-1 Appendix B Hematology Results ................................................................................................................. B-1 Appendix C Organ Weights and Organ-Weight-to-Body-Weight Ratios .................................................... C-1 Appendix D Reproductive Tissue Evaluations and Estrous Cycle Characterization ...................................D-1 Appendix E Genetic Toxicology ................................................................................................................. E-1 Appendix F Chemical Characterization and Dose Formulation Studies ..................................................... F-1 Appendix G Ingredients, Nutrient Composition, and Contaminant Levels in NTP-2000 Rat and Mouse Ration........................................................................................ G-1 Appendix H Sentinel Animal Program ........................................................................................................ H-1 6 Cedarwood Oil, NTP TOX 86 SUMMARY Background Virginia cedarwood oil is a widely used fragrance ingredient in many personal and household products such as soaps, cleaning products, and insecticides. We conducted short-term (3-month) tests to determine if there were any toxic effects of cedarwood oil on rats or mice. Because skin contact is common in humans, a dermal route of exposure was selected for these studies. Methods We applied solutions of cedarwood oil either neat or in ethanol to the skin on the backs of male and female rats and mice 5 days per week for 3 months. There were 10 rodents in each dose group. Doses were 6.25%, 12.5%, 25%, and 50% cedarwood oil in ethanol and 100% (neat) cedarwood oil. These doses corresponded to approximately 31.25 to 500 milligrams (mg) of cedarwood oil per kilogram (kg) of body weight in rats and 125 to 2,000 mg/kg in mice. One control group (vehicle control) received ethanol alone and served as the control group for dose groups that were dosed with 6.25% to 50% cedarwood oil in ethanol. Another control group (untreated control) received no administration of ethanol and served as the control group for the dose group exposed to 100% cedarwood oil. During the course of the studies, samples were collected for estrous cycle characterization. At the end of the studies, samples were collected for hematology, reproductive tissue evaluations, and genetic toxicology studies (mice only), and more than 40 tissues were collected from each animal for histopathology diagnosis. Results Survival was decreased in male and female mice dosed with 100% cedarwood oil. Mean body weights of animals exposed to cedarwood oil were decreased in the 50% and 100% groups of male and female rats and in the 25% and 50% groups of male and female mice. At the site of cedarwood oil application on the skin, there were several clinical observations (irritation, thicker skin, and ulcers) that corresponded with the occurrence of skin lesions (hyperplasia, hyperkeratosis, inflammation, and fibrosis). These skin lesions occurred in rats dosed with 12.5% to 100% cedarwood oil, in all dosed groups of mice, and were more severe in mice. Conclusions We conclude that dermal exposure to cedarwood oil for 3 months resulted in skin lesions at the site of cedarwood oil application in both rats and mice. 7 ABSTRACT VIRGINIA CEDARWOOD OIL CAS No. 8000-27-9 Synonyms: Cedar oil; cedarwood oil; oil of cedarwood; red cedarwood oil Virginia cedarwood oil (hereafter referred to as cedarwood oil) is extracted from Juniperus virginiana trees by steam distillation and contains cedrol, cedrene, cedrenol, cedral, cuperene, thujopsene, and widdrol as primary components. Cedarwood oil is used as a fragrance in cosmetic products, as a pesticide, and as a source material for production of other fragrance materials with cedarwood odors. Cedarwood oil was nominated for toxicity testing by the National Cancer Institute based on widespread and potentially increasing human exposure to the substance, and a lack of toxicology data. The dermal route of administration was selected for these studies because it is the most common route of exposure in humans due to its frequent use as a pesticide and as a fragrance in household products and cosmetics. Male and female F344/N rats and B6C3F1/N mice were administered cedarwood oil dermally for 3 months. Genetic toxicology studies were conducted in Salmonella typhimurium and mouse peripheral blood erythrocytes. Groups of 10 male and 10 female rats and mice received no treatment (untreated control) or were administered cedarwood oil in 95% aqueous ethanol dermally at concentrations of 0% (vehicle control), 6.25%, 12.5%, 25%, 8 Cedarwood Oil, NTP TOX 86 50%, or 100% (neat) 5 days per week for 14 weeks. Formulations were administered at a volume of 0.5 mL/kg body weight (rats) or 2.0 mL/kg (mice), which resulted in the mice receiving higher doses of cedarwood oil than rats in respect to the equivalent dose in mg/kg. Dose ranges for mice and rats were estimated as 125 to 2,000 mg cedarwood oil/kg body weight and 31.25 to 500 mg/kg, respectively. Statistical analyses of effects in dosed groups (except 100%) were conducted with comparisons to the appropriate vehicle control group; groups dosed with 100% cedarwood oil were compared to the appropriate untreated control group. With the exceptions of two males in the 100% group, all rats survived to the end of the study. Final mean body weights and body weight gains of 50% and 100% males and females were significantly less than those of their respective control groups, and the mean body weight gain of male rats in the 25% group was significantly less than that of the vehicle control group. Treatment-related clinical observations included irritation, thickening, and ulceration of the skin at the site of application in most males in the 25% or greater groups and most females in the 12.5% or greater groups. In rats, relative liver weights of males in the 50% and 100% groups, and absolute and relative liver weights of females in the 100% group were significantly greater than those in the respective control groups. The absolute kidney weight of 100% males and relative kidney weights of males in the 50% and 100% groups were significantly greater than those in the respective control groups. The absolute thymus weights of 25% and 100% males and the absolute and relative thymus weights of 100% females were significantly less than those in the respective control groups. Compared to those in the respective control groups, the incidences of several nonneoplastic lesions of the skin at the site of application were significantly increased in dosed groups of male and female rats. The incidences of epidermal hyperplasia were significantly increased in 12.5% or greater males and all dosed groups of females. The incidences of epidermal hyperkeratosis and sebaceous gland hyperplasia were significantly increased in 25% or greater males and 12.5% or greater females. The incidences of epidermal ulcer were significantly increased in the 50% and 100% groups of males and females. The incidences of chronic active inflammation were significantly increased in 12.5% or greater males and females. The incidences of dermal fibrosis and hair follicle hyperplasia were significantly increased in 25% or greater males and females. Hematology effects included increased leukon (white blood cell and differential counts) in 100% males and 50% or greater females and treatment-related decreases in erythron (hematocrit, hemoglobin, and erythrocyte counts) in 100% males and females. In the kidney of rats, the incidences of renal tubule degeneration were significantly increased in 50% and 100% males compared to those in the respective control groups. In addition, the incidences of renal tubule granular casts and hyaline droplet accumulation were significantly increased in males in the 25% or greater groups compared to those in the respective control groups; the severity of these lesions increased with increasing dose. In the bone marrow of rats, the incidences of hyperplasia were significantly increased in 100% males and females compared to those in the untreated control groups. Cedarwood Oil, NTP TOX 86 9 Due to the severities of skin lesions at the site of application, all male and female mice in the 100% groups, one male and one female each in the 50% groups, and one male in the 12.5% group were euthanized during weeks 10, 11, and 12, respectively; all other male and female mice survived to the end of the study. The final mean body weights of 25% and 50% males and 12.5% or greater females and the mean body weight gains of 12.5% or greater males and females were significantly less than those of the vehicle control groups. Test article-related clinical observations included irritation, thickening, and ulceration of the skin at the site of application in most dosed mice. In mice, the absolute liver weights of 50% males and females and relative liver weights of all dosed groups of males and females were significantly greater than those of the vehicle control groups. The absolute kidney weight of 50% females and relative kidney weights of 12.5% or greater females were significantly greater than those of the vehicle control groups. Absolute thymus weights of 25% and 50% males and 12.5% or greater females were significantly less than those of the vehicle controls. The incidences of epidermal hyperplasia, hyperkeratosis, and ulcer and chronic active inflammation, dermal fibrosis, hair follicle hyperplasia, and sebaceous gland hyperplasia were significantly increased in 12.5% or greater groups of male and female mice compared to those in the respective control groups. In addition, compared to the occurrences in the vehicle control groups, the incidences of epidermal hyperplasia and hyperkeratosis and chronic active inflammation, hair follicle hyperplasia, and sebaceous gland hyperplasia were significantly increased in 6.25% males, and the incidences of epidermal hyperplasia, chronic active inflammation, and sebaceous gland hyperplasia were significantly increased in 6.25% females. Hematology effects included increased leukon (white blood cells and differential counts) in 25% or greater females and treatment-related decreases in erythron (hematocrit, hemoglobin, and erythrocyte counts) in 12.5% or greater males and 25% and 50% females. In the liver, the incidences of hepatocyte glycogen depletion were significantly increased in males and females in the 12.5% or greater groups compared to those in the respective control groups. In the thymus, the incidences of atrophy were significantly increased in 25% or greater males compared to those in the respective controls. In the kidney, the incidence of nephropathy was significantly increased in 100% males compared to that in the untreated controls. Cedarwood oil was not mutagenic in S. typhimurium strains TA98, TA100, or TA102 with or without exogenous metabolic activation. No increase in micronucleated erythrocytes was seen in blood samples obtained from male B6C3F1/N mice treated with cedarwood oil for 3 months via dermal application; a small increase in micronucleated erythrocytes, judged to be equivocal, was seen in female B6C3F1/N mice, however. No significant alterations in the percentage of polychromatic erythrocytes (reticulocytes) were seen in male or female mice, suggesting that dermally-applied cedarwood oil did not induce bone marrow toxicity. 10 Cedarwood Oil, NTP TOX 86 Under the conditions of the 3-month dermal studies with Virginia cedarwood oil, there were treatment-related lesions in male and female rats and mice. Skin (at the site of application) and kidney were the major targets from administration of cedarwood oil in both rats and mice. Additionally, the liver and the thymus were considered secondary targets of cedarwood oil administration as a result of the skin effects at the site of application in both rats and mice. The most sensitive measures of cedarwood oil administration in each species and sex were: increased incidences of skin (site of application) lesions in male [lowest-observed-effect-level (LOEL) = 12.5%; approximately equivalent to 62.5 mg/kg] and female (LOEL = 6.25%; approximately equivalent to 31.25 mg/kg) rats and increased incidences of skin (site of application) lesions (LOEL = 6.25%; approximately equivalent to 124 mg/kg) in male and female mice. Cedarwood Oil, NTP TOX 86 11 Summary of Findings Considered to be Toxicologically Relevant in Rats and Mice Administered Cedarwood Oil Dermally for 3 Monthsa Male F344/N Rats Female F344/N Rats Male B6C3F1/N Mice Female B6C3F1/N Mice Concentrations by dermal application 0% (UC), 0% (VC), 6.25%, 12.5%, 25%, 50%, or 100% 0% (UC), 0% (VC), 6.25%, 12.5%, 25%, 50%, or 100% 0% (UC), 0% (VC), 6.25%, 12.5%, 25%, 50%, or 100% 0% (UC), 0% (VC), 6.25%, 12.5%, 25%, 50%, or 100% Survival rates 10/10, 10/10, 10/10, 10/10, 10/10, 10/10, 8/10 10/10, 10/10, 10/10, 10/10, 10/10, 10/10, 10/10 10/10, 10/10, 10/10, 9/10, 10/10, 9/10, 0/10c 10/10, 10/10, 10/10, 10/10, 10/10, 9/10, 0/10c Body weights 50% group 9% less than the vehicle control group; 100% group 14% less than the untreated control group 50% group 8% less than the vehicle control group; 100% group 11% less than the untreated control group 25% group 10% less than the vehicle control group; 50% group 14% less than the vehicle control group 12.5% group 12% less than the vehicle control group; 25% group 19% less than the vehicle control group; 50% group 13% less than the vehicle control group Clinical observations Irritation, thickening, and ulceration of the skin at the site of application Irritation, thickening, and ulceration of the skin at the site of application Irritation, thickening, and ulceration of the skin at the site of application Irritation, thickening, and ulceration of the skin at the site of application iver (absolute and relative) Thymus (absolute and relative) iver (absolute and relative) Thymus (absolute) Organ weights iver (relativeb) bsolute and relative) Thymus (absolute) iver (absolute and relative) bsolute and relative) Thymus (absolute) white blood cells Hematology Hematocrit Hemoglobin Erythrocytes Hematocrit Hemoglobin Erythrocytes Reproductive toxicity None None None None Nonneoplastic effects Skin (site of application): epidermis, hyperplasia (0/10, 0/10, 2/10, 4/10, 10/10, 10/10, 9/10); epidermis, hyperkeratosis (0/10, 0/10, 0/10, 1/10, 7/10, 10/10, 10/10); sebaceous gland, hyperplasia (0/10, 0/10, 0/10, 0/10, 9/10, 10/10, 10/10); epidermis, ulcer (0/10, 0/10, 0/10, 1/10, 1/10, 7/10, 8/10); inflammation, chronic active (0/10, 0/10, 1/10, 4/10, 9/10, 10/10, 10/10); dermis, fibrosis (0/10, 0/10, 0/10, 0/10, 7/10, 10/10, 10/10); hair follicle, hyperplasia (0/10, 0/10, 0/10, 0/10, 9/10, 10/10, 9/10) Skin (site of application): epidermis, hyperplasia (0/10, 0/10, 4/10, 7/10, 10/10, 10/10, 10/10); epidermis, hyperkeratosis (0/10, 0/10, 1/10, 5/10, 10/10, 10/10, 10/10); sebaceous gland, hyperplasia (0/10, 0/10, 0/10, 4/10, 9/10, 10/10, 10/10); epidermis, ulcer (0/10, 0/10, 0/10, 1/10, 1/10, 4/10, 10/10); inflammation, chronic active (0/10, 0/10, 0/10, 7/10, 10/10, 10/10, 10/10); dermis, fibrosis (0/10, 0/10, 0/10, 1/10, 7/10, 8/10, 10/10); hair follicle, hyperplasia (0/10, 0/10, 0/10, 1/10, 9/10, 10/10, 10/10) Skin (site of application): epidermis, hyperplasia (0/10, 0/9, 10/10, 10/10, 10/10, 10/10, 10/10); epidermis, hyperkeratosis (0/10, 0/9, 7/10, 10/10, 10/10, 10/10, 10/10); epidermis, ulcer (0/10, 0/9, 2/10, 8/10, 10/10, 9/10, 10/10); inflammation, chronic active (0/10, 0/9, 10/10, 10/10, 10/10, 10/10, 10/10); dermis, fibrosis (0/10, 0/9, 3/10, 10/10, 10/10, 10/10, 10/10); hair follicle, hyperplasia (0/10, 0/9, 10/10, 10/10, 10/10, 10/10, 10/10); sebaceous gland, hyperplasia (0/10, 0/9, 10/10, 10/10, 10/10, 10/10, 10/10) Skin (site of application): epidermis, hyperplasia (0/10, 0/10, 10/10, 10/10, 10/10, 10/10, 10/10); epidermis, hyperkeratosis (0/10, 0/10, 2/10, 10/10, 10/10, 10/10, 10/10 ); epidermis, ulcer (0/10, 0/10, 2/10, 6/10, 9/10, 10/10, 10/10); inflammation, chronic active (0/10, 0/10, 10/10, 10/10, 10/10, 10/10, 10/10); dermis, fibrosis (0/10, 0/10, 2/10, 10/10, 10/10, 10/10, 10/10); hair follicle, hyperplasia (0/10, 0/10, 3/10, 10/10, 10/10, 10/10, 10/10); sebaceous gland, hyperplasia (1/10, 0/10, 4/10, 10/10, 10/10, 10/10, 10/10) 12 Cedarwood Oil, NTP TOX 86 Summary of Findings Considered to be Toxicologically Relevant in Rats and Mice Administered Cedarwood Oil Dermally for 3 Months Male F344/N Rats Nonneoplastic effects (continued) Kidney: renal tubule, degeneration (2/10, 4/10, 5/10, 6/10, 7/10, 10/10, 10/10); renal tubule, casts granular (0/10, 0/10, 0/10, 0/10, 5/10, 10/10, 10/10); accumulation, hyaline droplet (0/10, 0/10, 0/10, 0/10, 10/10, 10/10, 10/10) Genetic toxicology Bacterial gene mutations: Micronucleated erythrocytes Mouse peripheral blood in vivo: a b c Female F344/N Rats Male B6C3F1/N Mice Female B6C3F1/N Mice Kidney: nephropathy (0/10, 2/10, 0/10, 1/10, 2/10, 3/10, 5/10) Negative in S. typhimurium strains TA98, TA100, and TA102 with and without S9 Negative in males and equivocal in females Reported treatment-related effects are in comparison to concurrent controls; untreated control (UC) compared to 100% group and ethanol vehicle control (VC) compared to all other dosed groups Relative to body weight Due to the severity of skin lesions, all 100% mice were euthanized during week 10 
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