TOX- 81: α-Pinene Administered by Inhalation to F344/N Rats and B6C3F1/N Mice

119 Pages·2016·5.72 MB·English

NTP T R
echNical ePoRT oN
T S
The oxiciTy TudieS of

α-P (caSRN 80-56-8)
iNeNe
a i
dmiNiSTeRed by NhalaTioN To
f344/N R
aTS aNd
b6c3f1/N m
ice
NTP TOX 81
MAY 2016
NTP Technical Report on the
Toxicity Studies of α-Pinene (CAS No. 80-56-8)
Administered by Inhalation to F344/N Rats and
B6C3F1/N Mice
Toxicity Report 81
May 2016
National Toxicology Program
Public Health Service
U.S. Department of Health and Human Services
ISSN: 2378-8992

Research Triangle Park, North Carolina, USA
α-Pinene, NTP TOX 81
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 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 National Center for Biotechnology Information
(NCBI) Bookshelf database and are available free of charge electronically on the NTP website
(http://ntp.niehs.nih.gov).
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α-Pinene, NTP TOX 81
Table of Contents
Foreword ......................................................................................................................................... ii
Tables ............................................................................................................................................. iv
Figures..............................................................................................................................................v
About This Report.......................................................................................................................... vi
Peer Review ................................................................................................................................. viii
Publication Details ......................................................................................................................... ix
Abstract ............................................................................................................................................x
Introduction ......................................................................................................................................1
Chemical and Physical Properties ...............................................................................................1
Production, Use, and Human Exposure ......................................................................................1
Absorption, Distribution, Metabolism, Excretion, and Toxicokinetics ......................................2
Toxicity .......................................................................................................................................3
Carcinogenicity ...........................................................................................................................3
Genetic Toxicity ..........................................................................................................................4
Study Rationale ...........................................................................................................................4
Materials and Methods .....................................................................................................................6
Procurement and Characterization of α-Pinene ..........................................................................6
Vapor Generation and Exposure System ....................................................................................6
Vapor Concentration Monitoring ................................................................................................7
Chamber Atmosphere Characterization ......................................................................................7
Animal Source .............................................................................................................................7
Two-week Studies .......................................................................................................................8
Three-month Studies ...................................................................................................................8
Statistical Methods ....................................................................................................................13
Calculation and Analysis of Lesion Incidences .................................................................13
Analysis of Continuous Variables .....................................................................................13
Quality Assurance Methods ......................................................................................................14
Genetic Toxicology ...................................................................................................................14
Bacterial Mutagenicity Test Protocol ................................................................................14
Mouse Peripheral Blood Micronucleus Test Protocol .......................................................14
Evaluation Protocol ............................................................................................................15
Results ............................................................................................................................................16
Rats ............................................................................................................................................16
Two-week Study ................................................................................................................16
Three-month Study ............................................................................................................17
Mice...........................................................................................................................................23
Two-week Study ................................................................................................................23
Three-month Study ............................................................................................................24
Genetic Toxicology ...................................................................................................................28
Discussion ......................................................................................................................................34
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α-Pinene, NTP TOX 81
Errata ..............................................................................................................................................38
References ......................................................................................................................................39
Appendix A. Summary of Neoplasms and Nonneoplastic Lesions in Rats and Mice ................ A-1
Appendix B. Genetic Toxicology ................................................................................................B-1
Appendix C. Clinical Pathology Results .....................................................................................C-1
Appendix D. Organ Weights and Organ-Weight-to-Body Ratios .............................................. D-1
Appendix E. Reproductive Tissue Evaluations and Estrous Cycle Characterization .................. E-1
Appendix F. Chemical Characterization and Generation of Chamber Concentrations ............... 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

Tables
Summary of Findings Considered to be Toxicologically Relevant in Rats and Mice
Exposed to α-Pinene by Inhalation for Three Months .................................................... xi
Table 1. Experimental Design and Materials and Methods in the Inhalation Studies of α-
Pinene ..............................................................................................................................10
Table 2. Survival and Body Weights of Rats in the Two-week Inhalation Study of α-
Pinene ..............................................................................................................................16
Table 3. Survival and Body Weights of Rats in the Three-month Inhalation Study of α-
Pinene ..............................................................................................................................18
Table 4. Selected Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats in
the Three-month Inhalation Study of α-Pinene ...............................................................21
Table 5. Epididymal Spermatozoal Measurements for Male Rats in the Three-month
Inhalation Study of α-Pinene ...........................................................................................22
Table 6. Incidences of Nonneoplastic Lesions of the Kidney in Male Rats in the Three-
month Inhalation Study of α-Pinene ................................................................................23
Table 7. Survival and Body Weights of Mice in the Two-week Inhalation Study of α-
Pinene ..............................................................................................................................23
Table 8. Survival and Body Weights of Mice in the Three-month Inhalation Study of α-
Pinene ..............................................................................................................................24
Table 9. Selected Organ Weights and Organ-Weight-to-Body-Weight Ratios for Mice in
the Three-month Inhalation Study of α-Pinene ...............................................................26
Table 10. Epididymal Spermatozoal Measurements of Male Mice in the Three-month
Inhalation Study of α-Pinenea ........................................................................................27
Table 11. Incidences of Nonneoplastic Lesions of the Urinary Bladder in Mice in the
Three-month Inhalation Study of α-Pinene ...................................................................28

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α-Pinene, NTP TOX 81
Figures
Figure 1. α-Pinene (CAS No. 80-56-8; Chemical Formula: C H ; Molecular Weight:
10 16
136.24) ..............................................................................................................................1
Figure 2. Growth Curves for Rats Exposed to α-Pinene by Inhalation for Three Months ............19
Figure 3. Growth Curves for Mice Exposed to α-Pinene by Inhalation for Three Months ...........25
Figure 4. Kidney of a Chamber Control Male F344/N Rat in the Three-month Inhalation
Study of α-Pinene (H&E) ...............................................................................................29
Figure 5. Higher Magnification of Figure 4 (H&E) ......................................................................29
Figure 6. Kidney of a Male F344/N Rat Exposed to 400 ppm α-Pinene by Inhalation for
Three Months (H&E) .....................................................................................................29
Figure 7. Higher Magnification of Figure 6 (H&E) ......................................................................30
Figure 8. Kidney of a Chamber Control Male F344/N Rat in the Three-month Inhalation
Study of α-Pinene (H&E) ...............................................................................................30
Figure 9. Kidney of a Chamber Control Male F344/N Rat in the Three-month Inhalation
Study of α-Pinene (Mallory-Heidenhain) .......................................................................30
Figure 10. α2µ Globulin Nephropathy in the Kidney of a Male F344/N Rat Exposed to
400 ppm α-Pinene by Inhalation for Three Months (H&E) .........................................31
Figure 11. α2µ Globulin Nephropathy in the Kidney of a Male F344/N Rat Exposed to
400 ppm α-Pinene by Inhalation for Three Months (Mallory-Heidenhain) .................31
Figure 12. Urinary Bladder of a Chamber Control Female B6C3F1/N Mouse in the
Three-month Inhalation Study of α-Pinene (H&E) ......................................................32
Figure 13. Higher Magnification of Figure 12 Showing Normal Transitional Epithelium
(Arrow) (H&E) .............................................................................................................32
Figure 14. Urinary Bladder of a Female B6C3F1/N Mouse Exposed to 400 ppm α-Pinene
by Inhalation for Three Months (H&E) .......................................................................33
Figure 15. Higher Magnification of Figure 14 Showing the Hyperplastic Transitional
Epithelium (Arrow) (H&E) ..........................................................................................33
This report has been reformatted to meet new NTP publishing requirements;
its content has not changed.
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α-Pinene, NTP TOX 81
About This Report
National Toxicology Program1
1Division of the National Toxicology Program, National Institute of Environmental Health
Sciences, Research Triangle Park, North Carolina, USA
Collaborators
C.V. Rider, R.A. Herbert, B. Atkinson, C.R. Blystone, M.C. Cora, J.A. Dill, P.M. Foster, S.L.
Grumbein, S.J. Harbo, B.K. Hayden, M.J. Hooth, C.D. Houle, A.P. King-Herbert, G.E. Kissling,
W.G. Lieuallen, D.E. Malarkey, R.A. Miller, B.J.T. Muir, S.L. Smith-Roe, M.D. Stout, G.S.
Travlos, S. Waidyanatha, N.J. Walker, Y. Wang, K.L. Witt
Division of the National Toxicology Program, National Institute of Environmental Health
Sciences, Research Triangle Park, North Carolina, USA
Evaluated and interpreted results and reported findings
C.V. Rider, Ph.D., Study Scientist
R.A. Herbert, D.V.M., Ph.D., Study Pathologist
C.R. Blystone, Ph.D.
M.C. Cora, D.V.M., Ph.D.
P.M. Foster, Ph.D.
M.J. Hooth, Ph.D.
A.P. King-Herbert, D.V.M.
G.E. Kissling, Ph.D.
D.E. Malarkey, D.V.M., Ph.D.
S.L. Smith-Roe, Ph.D.
M.D. Stout, Ph.D.
G.S. Travlos, D.V.M.
S. Waidyanatha, Ph.D.
N.J. Walker, Ph.D.
K.L. Witt, M.S.
Battelle Toxicology Northwest, Richland, Washington, USA
Conducted studies and evaluated pathology findings
J.A. Dill, Ph.D., Principal Investigator
S.L. Grumbein, D.V.M., Ph.D.
S.J. Harbo, D.V.M.
B.K. Hayden
Experimental Pathology Laboratories, Inc., Research Triangle Park, North Carolina, USA
Conducted pathology review
C.D. Houle, D.V.M.
R.A. Miller, D.V.M., Ph.D.
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α-Pinene, NTP TOX 81
Pathology Associates, a Division of Charles River Laboratories, Inc., Research Triangle
Park, North Carolina, USA
Coordinated NTP Pathology Peer Review (June 30, 2006)
W.G. Lieuallen, D.V.M., Ph.D.
Gene Logic Laboratories, Inc., Gaithersburg, Maryland, USA
Provided SMVCE analysis
B.J.T. Muir, Ph.D., Principal Investigator
B. Atkinson, M.Sc.
Y. Wang, M.S.
Contributors
NTP Pathology Peer Review, National Institute of Environmental Health Sciences,
Research Triangle Park, North Carolina, USA
Participated in NTP Pathology Peer Review (June 30, 2006)
J.P. Morrison, D.V.M., Pathology Associates, a Division of Charles River Laboratories, Inc.
R.A. Herbert, D.V.M., Ph.D., National Toxicology Program
Experimental Pathology Laboratories, Inc., Research Triangle Park, North Carolina, USA
Supervised pathology review
M.H. Hamlin, II, D.V.M., Principal Investigator
Dynamac Corporation, Research Triangle Park, North Carolina, USA
Prepared quality assessment audits
S. Brecher, Ph.D., Principal Investigator
S. Iyer, B.S.
V.S. Tharakan, D.V.M.
SRA International, Research Triangle Park, North Carolina, USA
Provided statistical analyses
R.W. Morris, Ph.D., Principal Investigator
L.J. Betz, M.S.
S.F. Harris, B.S.
Biotechnical Services, Inc., Little Rock, Arkansas, USA
Prepared Toxicity Study Report
S.R. Gunnels, M.A., Principal Investigator
B.F. Hall, M.S.
L.M. Harper, B.S.
P.C. Nader, B.S.E.
D.C. Serbus, Ph.D.
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α-Pinene, NTP TOX 81
Peer Review
The draft NTP Technical Report on the Toxicity Studies of α-Pinene (CAS No. 80-56-8)
Administered by Inhalation to F344/N Rats and B6C3F1/N Mice was evaluated by the reviewers
listed below. These reviewers served as independent scientists, not as representatives of any
institution, company, or governmental agency. In this capacity, reviewers determined if the
design and conditions of these NTP studies were appropriate and ensured that this NTP Toxicity
Study Report presents the experimental results and conclusions fully and clearly.
Peer Reviewers
Terry Gordon, Ph.D.
New York University School of Medicine
Tuxedo, New York, USA
Michael V. Pino, D.V.M., Ph.D.
Veterinary Toxicologic Pathology and Preclinical Drug Development
Albuquerque, New Mexico, USA
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α-Pinene, NTP TOX 81
Publication Details
Publisher: National Toxicology Program
Publishing Location: Research Triangle Park, NC
ISSN: 2378-8992
DOI: https://doi.org/10.22427/NTP-TOX-81
Report Series: NTP Toxicity Report Series
Report Series Number: 81
Official citation: National Toxicology Program (NTP). 2016. NTP technical report on the
toxicity studies of α-pinene (CAS no. 80-56-8) administered by inhalation to F344/N rats and
B6C3F1/N mice. Research Triangle Park, NC: National Toxicology Program. Toxicity
Report 81.
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