TRIM VX Administered by Inhalation to Wistar Han

198 Pages·2016·16.82 MB·English

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NTP TR 591
NOVEMBER 2016
NTP Technical Report on the
Toxicology and Carcinogenesis Studies of
TRIM® VX in Wistar Han [Crl:WI (Han)] Rats and
B6C3F1/N Mice (Inhalation Studies)
Technical Report 591
November 2016
National Toxicology Program
Public Health Service
U.S. Department of Health and Human Services
ISSN: 2378-8925
Research Triangle Park, North Carolina, USA
TRIM® VX, NTP TR 591
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, 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 Technical Report series began in 1976 with carcinogenesis studies conducted by the
National Cancer Institute. In 1981, this bioassay program was transferred to NTP. The studies
described in the Technical Report series are designed and conducted to characterize and evaluate
the toxicologic potential, including carcinogenic activity, of selected substances in laboratory
animals (usually two species, rats and mice). Substances selected for NTP toxicity and
carcinogenicity studies are chosen primarily on the basis of human exposure, level of production,
and chemical structure. The interpretive conclusions presented in NTP Technical 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 carcinogenic potential.
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 Technical Reports are available free of charge on the NTP website and cataloged in
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the National Institutes of Health). Data for these studies are included in NTP’s Chemical Effects
in Biological Systems database.
For questions about the reports and studies, please email NTP or call 984-287-3211.
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TRIM® VX, NTP TR 591
Table of Contents
Foreword ......................................................................................................................................... ii
Tables ...............................................................................................................................................v
Figures..............................................................................................................................................v
About This Report........................................................................................................................ viii
Explanation of Levels of Evidence of Carcinogenic Activity ....................................................... xi
Peer Review ................................................................................................................................. xiii
Publication Details ....................................................................................................................... xiv
Abstract ..........................................................................................................................................xv
TRIM® VX ................................................................................................................................xv
Three-month Study in Rats .......................................................................................................xv
Three-month Study in Mice ......................................................................................................xv
Two-year Study in Rats ........................................................................................................... xvi
Two-year Study in Mice.......................................................................................................... xvi
Genetic Toxicology ................................................................................................................ xvii
Conclusions ............................................................................................................................ xvii
Overview ...................................................................................................................................... xxi
Introduction ......................................................................................................................................1
Chemical and Physical Properties ...............................................................................................1
Production, Use, and Human Exposure ......................................................................................2
Regulatory Status ........................................................................................................................3
Absorption, Distribution, Metabolism, and Excretion ................................................................3
Toxicity .......................................................................................................................................3
Experimental Animals .........................................................................................................3
Humans ................................................................................................................................5
Reproductive and Developmental Toxicity ................................................................................6
Carcinogenicity ...........................................................................................................................6
Experimental Animals .........................................................................................................6
Humans ................................................................................................................................7
Genetic Toxicity ..........................................................................................................................8
Study Rationale ...........................................................................................................................9
Materials and Methods ...................................................................................................................10
Procurement and Characterization of TRIM® VX ....................................................................10
Aerosol Generation and Exposure System ................................................................................11
Aerosol Concentration Monitoring ...........................................................................................12
Chamber Atmosphere Characterization ....................................................................................12
Animal Source ...........................................................................................................................13
Animal Welfare .........................................................................................................................13
Three-month Studies .................................................................................................................13
Two-year Studies ......................................................................................................................15
Study Design ......................................................................................................................15
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TRIM® VX, NTP TR 591
Clinical Examinations and Pathology ................................................................................15
Statistical Methods ....................................................................................................................19
Survival Analyses ..............................................................................................................19
Calculation of Incidence ....................................................................................................19
Analysis of Neoplasm and Nonneoplastic Lesion Incidences ...........................................20
Analysis of Continuous Variables .....................................................................................20
Historical Control Data ......................................................................................................20
Quality Assurance Methods ......................................................................................................21
Genetic Toxicology ...................................................................................................................21
Results ............................................................................................................................................23
Data Availability .......................................................................................................................23
Rats ............................................................................................................................................23
Three-month Study ............................................................................................................23
Two-year Study ..................................................................................................................28
Mice ..........................................................................................................................................40
Three-month Study ............................................................................................................40
Two-year Study ..................................................................................................................46
Genetic Toxicology ...................................................................................................................57
Discussion ......................................................................................................................................71
Conclusions ....................................................................................................................................78
References ......................................................................................................................................79
Summary of Lesions in Male Rats in the Two-year Inhalation Study of
TRIM VX .............................................................................................................. A-1
Summary of Lesions in Female Rats in the Two-year Inhalation Study of
TRIM VX ...............................................................................................................B-1
Summary of Lesions in Male Mice in the Two-year Inhalation Study of
TRIM VX ...............................................................................................................C-1
Summary of Lesions in Female Mice in the Two-year Inhalation Study of
TRIM VX .............................................................................................................. D-1
Genetic Toxicology ................................................................................................ E-1
Clinical Pathology Results ...................................................................................... F-1
Organ Weights and Organ-Weight-to-Body-Weight Ratios ................................. G-1
Chemical Characterization and Generation of Chamber Concentrations ............. H-1
Ingredients, Nutrient Composition, and Contaminant Levels in NTP-2000
Rat and Mouse Ration ............................................................................................. I-1
Sentinel Animal Program ......................................................................................... J-1
Summary of Peer Review Panel Comments of Draft NTP Technical Report
on TRIM VX (TR 591) ......................................................................................... K-1
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TRIM® VX, NTP TR 591
Tables
Summary of the Two-year Carcinogenesis and Genetic Toxicology Studies of TRIM VX ..... xviii
Table 1. Measured Components of the Two Lots of TRIM VX Used in the Inhalation
Studies of TRIM VX .......................................................................................................11
Table 2. Experimental Design and Materials and Methods in the Inhalation Studies of
TRIM VX ........................................................................................................................16
Table 3. Survival and Body Weights of Rats in the Three-month Inhalation Study of
TRIM VX ........................................................................................................................23
Table 4. Selected Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats in
the Three-month Inhalation Study of TRIM VX .............................................................25
Table 5. Incidences of Nonneoplastic Lesions of the Respiratory System in Rats in the
Three-month Inhalation Study of TRIM VX ...................................................................26
Table 6. Survival of Rats in the Two-year Inhalation Study of TRIM VX ...................................29
Table 7. Mean Body Weights and Survival of Male Rats in the Two-year Inhalation
Study of TRIM VX ..........................................................................................................31
Table 8. Mean Body Weights and Survival of Female Rats in the Two-year Inhalation
Study of TRIM VX ..........................................................................................................32
Table 9. Incidences of Neoplasms and Nonneoplastic Lesions of the Respiratory System
in Rats in the Two-year Inhalation Study of TRIM VX ..................................................35
Table 10. Survival and Body Weights of Mice in the Three-month Inhalation Study of
TRIM VX ......................................................................................................................41
Table 11. Selected Organ Weights and Organ-Weight-to-Body-Weight Ratios for Mice in
the Three-month Inhalation Study of TRIM VX ..........................................................43
Table 12. Incidences of Nonneoplastic Lesions of the Respiratory System in Mice in the
Three-month Inhalation Study of TRIM VX ................................................................44
Table 13. Survival of Mice in the Two-year Inhalation Study of TRIM VX ................................46
Table 14. Mean Body Weights and Survival of Male Mice in the Two-year Inhalation
Study of TRIM VX .......................................................................................................49
Table 15. Mean Body Weights and Survival of Female Mice in the Two-year Inhalation
Study of TRIM VX .......................................................................................................50
Table 16. Incidences of Neoplasms and Nonneoplastic Lesions of the Respiratory System
in Mice in the Two-year Inhalation Study of TRIM VX ..............................................53

Figures
Figure 1. Growth Curves for Rats Exposed to TRIM VX by Inhalation for Three Months ..........24
Figure 2. Kaplan-Meier Survival Curves for Rats Exposed to TRIM VX by Inhalation for
Two Years ......................................................................................................................30
Figure 3. Growth Curves for Rats Exposed to TRIM VX by Inhalation for Two Years ...............33
Figure 4. Growth Curves for Mice Exposed to TRIM VX by Inhalation for Three Months .........42
Figure 5. Kaplan-Meier Survival Curves for Mice Exposed to TRIM VX by Inhalation for
Two Years ......................................................................................................................48
Figure 6. Growth Curves for Mice Exposed to TRIM VX by Inhalation for 2 Years ...................51
Figure 7. Alveolar/Bronchiolar Adenoma in the Lung of a Female Wistar Han Rat
Exposed to 100 mg/m3 TRIM VX by Inhalation for Two Years (H&E) .......................58
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TRIM® VX, NTP TR 591
Figure 8. Higher Magnification of Figure 7 (H&E) ......................................................................58
Figure 9. Alveolar/Bronchiolar Carcinoma in the Lung of a Male Wistar Han Rat
Exposed to 100 mg/m3 TRIM VX by Inhalation for Two Years (H&E) .......................58
Figure 10. Higher Magnification of Figure 9 (H&E) ....................................................................59
Figure 11. Cystic Keratinizing Epithelioma in the Lung of a Female Wistar Han Rat
Exposed to 100 mg/m3 TRIM VX by Inhalation for Two Years (H&E) .....................59
Figure 12. Higher Magnification of Figure 11 (H&E) ..................................................................59
Figure 13. Discrete Area of Alveolar Epithelium Hyperplasia (Arrows) in the Lung of a
Female Wistar Han Rat Exposed to 100 mg/m3 TRIM VX by Inhalation for
Two Years (H&E) ........................................................................................................60
Figure 14. Higher Magnification of Figure 13 (H&E) ..................................................................60
Figure 15. Multifocal Areas of Alveolar/Bronchiolar Epithelium Hyperplasia (arrows) in
the Lung of a Female Wistar Han Rat Exposed to 100 mg/m3 TRIM VX by
Inhalation for Two Years (H&E) .................................................................................60
Figure 16. Higher Magnification of Figure 15 (H&E) ..................................................................61
Figure 17. Focal Area of Alveolar Epithelium Squamous Metaplasia (Long Arrows)
Associated with Alveolar Epithelium Hyperplasia (Short Arrows) in the Lung
of a Female Wistar Han Rat Exposed to 100 mg/m3 TRIM VX by Inhalation
for Two Years (H&E) ..................................................................................................61
Figure 18. Higher Magnification of Figure 17 (H&E) ..................................................................61
Figure 19. Infiltration Cellular, Histiocyte (Arrows) within an Area of Alveolar
Epithelium Hyperplasia in the Lung of a Male Wistar Han Rat Exposed to
30 mg/m3 TRIM VX by Inhalation for Two Years (H&E) .........................................62
Figure 20. Higher Magnification of Figure 19 (H&E) ..................................................................62
Figure 21. Alveolus Proteinosis in the Lung of a Female Wistar Han Rat Exposed to
100 mg/m3 TRIM VX by Inhalation for Two Years (H&E) .......................................62
Figure 22. Alveolus Proteinosis in the Lung of a Female Wistar Han Rat Exposed to
100 mg/m3 TRIM VX by Inhalation for 2 Years (H&E) .............................................63
Figure 23. An Area of Marked Chronic Active Inflammation (Long Arrows) in the Lung
of a Male Wistar Han Rat Exposed to 100 mg/m3 TRIM VX by Inhalation for
Two Years (H&E) ........................................................................................................63
Figure 24. Higher Magnification of Figure 23 (H&E) ..................................................................63
Figure 25. Section of the Lung of a Female Wistar Han Rat Exposed to 100 mg/m3
TRIM VX by Inhalation for Two Years (Oil-red-O) ...................................................64
Figure 26. Lymphohistiocytic Hyperplasia in the Bronchus-Associated Lymphoid Tissue
(BALT) in the Lung of a Female Wistar Han Rat Exposed to 100 mg/m3
TRIM VX by Inhalation for Two Years (H&E) ..........................................................64
Figure 27. Higher Magnification of Figure 26 (H&E) ..................................................................64
Figure 28. Suppurative Inflammation in a Nasal Maxilloturbinate in the Level II Section
of the Nose of a Female Wistar Han Rat Exposed to 100 mg/m3 TRIM VX by
Inhalation for 2 Years (H&E) ......................................................................................65
Figure 29. Focal Area of Respiratory Epithelium Hyperplasia at the Tip of a
Maxilloturbinate in the Nose of a Female Wistar Han Rat Exposed to
100 mg/m3 TRIM VX by Inhalation for Two Years (H&E) .......................................65
Figure 30. Transitional Epithelium Hyperplasia in the Nose of a Female Wistar Han Rat
Exposed to 100 mg/m3 TRIM VX by Inhalation for Two Years (H&E) .....................65
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TRIM® VX, NTP TR 591
Figure 31. Hyaline Droplet Accumulation in the Olfactory Epithelium in the Nose of a
Male Wistar Han Rat Exposed to 100 mg/m3 TRIM VX by Inhalation for
Two Years (H&E) ........................................................................................................66
Figure 32. Hyperplasia of the Submucosal Glands in the Olfactory Epithelium of the
Ventral Ethmoid Turbinates in the Nose of a Male Wistar Han Rat Exposed to
100 mg/m3 TRIM VX by Inhalation for Two Years (H&E) .......................................66
Figure 33. Squamous Metaplasia in the Larynx of a Male Wistar Han Rat Exposed to
100 mg/m3 TRIM VX by Inhalation for 2 Years (H&E) .............................................66
Figure 34. Lymphohistiocytic Hyperplasia in the Bronchial Lymph Node of a Male
Wistar Han Rat Exposed to 100 mg/m3 TRIM VX by Inhalation for Two
Years (H&E) ................................................................................................................67
Figure 35. Alveolar/Bronchiolar Adenoma in the Lung of a Female B6C3F1/N Mouse
Exposed to 100 mg/m3 TRIM VX by Inhalation for Two Years (H&E) .....................67
Figure 36. Higher Magnification of Figure 35 (H&E) ..................................................................67
Figure 37. Alveolar/Bronchiolar Carcinoma in the Lung of a Male B6C3F1/N Mouse
Exposed to 100 mg/m3 TRIM VX by Inhalation for 2 Years (H&E) ..........................68
Figure 38. Higher Magnification of Figure 37 (H&E) ..................................................................68
Figure 39. Bronchiole Epithelium Adenoma in the Lung of a Male B6C3F1/N Mouse
Exposed to 30 mg/m3 TRIM VX by Inhalation for Two Years (H&E) .......................68
Figure 40. Higher Magnification of Figure 39 (H&E) ..................................................................69
Figure 41. Focal Alveolar Epithelium Hyperplasia in the Lung of a Male B6C3F1/N
Mouse Exposed to 100 mg/m3 TRIM VX by Inhalation for Two Years (H&E) .........69
Figure 42. Higher Magnification of Figure 41 (H&E) ..................................................................69
Figure 43. Alveolar/Bronchiolar Epithelium Hyperplasia in the Lung of a Male
B6C3F1/N Mouse Exposed to 100 mg/m3 TRIM VX by Inhalation for Two
Years (H&E) ................................................................................................................70
Figure 44. Higher Magnification of Figure 43 (H&E) ..................................................................70
Figure 45. Exudate in the Ventral Nasal Passages in the Ethmoid Region of the Nose of a
Female B6C3F1/N Mouse Exposed to 100 mg/m3 TRIM VX by Inhalation for
Two Years (H&E) ........................................................................................................70

This report has been reformatted to meet new NTP publishing requirements;
its content has not changed.
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TRIM® VX, NTP TR 591
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
K.R. Ryan, R.A. Herbert, K.S. Janardhan, C. R. Blystone, M.C. Cora, T. Crabbs, J.A. Dill, P.M.
Foster, M.M. Gruebbel, S.L. Grumbein, B.K. Hayden, G.D. Hill, M.J. Hooth, A.P. King-Herbert,
G.E. Kissling, D.E. Malarkey, D.L. Morgan, K.M. Patton, M.J. Ryan, S.L. Smith-Roe, L.M.
Staska, M.D. Stout, G.S. Travlos, M.K. Vallant, S. Waidyanatha, N.J. Walker, 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
K.R. Ryan, Ph.D., Study Scientist
R.A. Herbert, D.V.M., Ph.D., Co-Study Pathologist
C.R. Blystone, Ph.D.
M.C. Cora, D.V.M.
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.
D.L. Morgan, Ph.D.
S.L. Smith-Roe, Ph.D.
M.D. Stout, Ph.D.
G.S. Travlos, D.V.M.
M.K. Vallant, M.S., M.T.
S. Waidyanatha, Ph.D.
N.J. Walker, Ph.D.
K.L. Witt, M.S.
ILS, Inc., Research Triangle Park, North Carolina, USA
Evaluated and interpreted results and reported findings
K.S. Janardhan, Ph.D., Co-Study Pathologist

Coordinated NTP Pathology Working Groups
G.D. Hill, D.V.M., Ph.D., Coordinated 2-year rats (February 20, 2014)
L.M. Staska, D.V.M., Ph.D., Coordinated 2-year mice (May 6, 2014)
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.
B.K. Hayden
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TRIM® VX, NTP TR 591
K.M. Patton, D.V.M., Ph.D.
M.J. Ryan, D.V.M., Ph.D.
L.M. Staska, D.V.M., Ph.D.
Experimental Pathology Laboratories, Inc., Research Triangle Park, North Carolina, USA
Conducted pathology review
T. Crabbs, D.V.M.
M.M. Gruebbel, D.V.M., Ph.D.
Contributors
Experimental Pathology Laboratories, Inc., Research Triangle Park, North Carolina, USA
Supervised pathology review
M.H. Hamlin, II, D.V.M., Principal Investigator
NTP Pathology Working Group, National Institute of Environmental Health Sciences,
Research Triangle Park, North Carolina, USA
Participated in NTP Pathology Working Group on 2-year rats (February 20, 2014)
M.F. Cesta, D.V.M., Ph.D., National Toxicology Program
S.A. Elmore, D.V.M., M.S., 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
D.E. Malarkey, D.V.M., Ph.D, National Toxicology Program
T. Osborne, D.V.M., Ph.D., National Toxicology Program
M.V. Pino, D.V.M., Ph.D., Sanofi-aventis
D.B. Rao, Ph.D., ILS, Inc.
L.M. Staska, D.V.M., Ph.D., ILS, Inc.

Participated in NTP Pathology Working Group on 2-year mice (May 6, 2014)
M.F. Cesta, D.V.M., Ph.D., National Toxicology Program
T. Crabbs, D.V.M., Experimental Pathology Laboratories, 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
G.D. Hill, D.V.M., Ph.D., ILS, Inc.
K.S. Janardhan, Ph.D., ILS, Inc.
D.B. Rao, Ph.D., ILS, Inc.
R.A. Renne, D.V.M., ToxPath Consulting
Social & Scientific Systems, Inc., Research Triangle Park, North Carolina, USA
Provided statistical analyses
M.V. Smith, Ph.D., Principal Investigator
L.J. Betz, M.S.
S.F. Harris, B.S.
Dynamac Corporation, Research Triangle Park, North Carolina, USA
Prepared quality assessment audits
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