National Toxicology Program

Toxicity Report Series

Number 57

NTP Technical Report

on the Toxicity Studies of

Benzyltrimethylammonium Chloride

(CAS No. 56-93-9)

Administered by Gavage

to F344/N Rats and B6C3F1 Mice

February 2000

NIH Publication No. 00-4405

U.S. Department of Health and Human Services

Public Health Service

National Institutes of Health
FOREWORD

The National Toxicology Program (NTP) is made up of four charter agencies of the U.S. Department of

Health and Human Services (DHHS): the National Cancer Institute (NCI), National Institutes of Health;

the National Institute of Environmental Health Sciences (NIEHS), National Institutes of Health; the

National Center for Toxicological Research (NCTR), Food and Drug Administration; and the National

Institute for Occupational Safety and Health (NIOSH), Centers for Disease Control and Prevention. In

July 1981, the Carcinogenesis Bioassay Testing Program, NCI, was transferred to the NIEHS. The NTP

coordinates the relevant programs, staff, and resources from these Public Health Service agencies relating

to basic and applied research and to biological assay development and validation.

The NTP develops, evaluates, and disseminates scientific information about potentially toxic and hazardous

chemicals. This knowledge is used for protecting the health of the American people and for the primary

prevention of disease.

The studies described in this Toxicity Study Report were performed under the direction of the NIEHS and

were conducted in compliance with NTP laboratory health and safety requirements and must meet or

exceed all applicable federal, state, and local health and safety regulations. Animal care and use were in

accordance with the Public Health Service Policy on Humane Care and Use of Animals.

These studies are designed and conducted to characterize and evaluate the toxicologic potential of selected

chemicals in laboratory animals (usually two species, rats and mice). Chemicals selected for NTP

toxicology studies are chosen primarily on the bases of human exposure, level of production, and chemical

structure. The interpretive conclusions presented in this Toxicity Study Report are based only on the

results of these NTP studies. Extrapolation of these results to other species and quantitative risk analyses

for humans require wider analyses beyond the purview of these studies. Selection per se is not an indicator

of a chemicalís toxic potential.

Listings of all published NTP reports and ongoing studies are available from NTP Central Data

Management, NIEHS, P.O. Box 12233, MD E1-02, Research Triangle Park, NC 27709 (919-541-3419). 

Other information about NTP studies is available at the NTPís World Wide Web site: 

http://ntp-server.niehs.nih.gov.
National Toxicology Program

Toxicity Report Series

Number 57

NTP Technical Report

on the Toxicity Studies of

Benzyltrimethylammonium Chloride

(CAS No. 56-93-9)

Administered by Gavage

to F344/N Rats and B6C3F1 Mice

Kamal M. Abdo, Ph.D., Study Scientist

National Toxicology Program

P.O. Box 12233

Research Triangle Park, NC 27709

U.S. Department of Health and Human Services

Public Health Service

National Institutes of Health
2

CONTRIBUTORS

National Toxicology Program

Evaluated and interpreted results and report findings

K.M. Abdo, Ph.D., Study Scientist

J.R. Bucher, Ph.D.

R.E. Chapin, Ph.D.

G.J. Harry, Ph.D.

J. Mahler, D.V.M.

C.S. Smith, Ph.D.

G.S. Travlos, D.V.M.

K.L. Witt, M.S., Integrated Laboratory Systems, Inc.

Microbiological Associates, Inc.

Conducted studies

M.L. Wenk, Ph.D., Principal Investigator

J.M. Pletcher, D.V.M.

Experimental Pathology Laboratories, Inc.

Provided pathology quality assurance

J.F. Hardisty, D.V.M., Principal Investigator

S. Botts, D.V.M, M.S., Ph.D.

Pathology Associates International

Evaluated pathology findings

L.H. Brennecke, D.V.M.

R.M. Kovatch, D.V.M.

NTP Pathology Review

Evaluated slides and prepared pathology report (22 November 1996)

J.C. Seely, D.V.M., Chairperson

PATHCO, Inc.

J. Mahler, D.V.M.

National Toxicology Program

Analytical Sciences, Inc.

Provided statistical analyses

R.W. Morris, M.S., Principal Investigator

K.P. McGowan, M.B.A.

M.A. Mauney, M.S.

N.G. Mintz, B.S.

J.T. Scott, M.S.

Biotechnical Services, Inc.

Prepared Toxicity Study Report

S.R. Gunnels, M.A., Principal Investigator

A.M. Macri-Hanson, M.A., M.F.A.

M.L. Rainer, B.S.

W.D. Sharp, B.A., B.S.

D.P. Shaw, B.A.
3

PEER REVIEW

The draft report on the toxicity studies of benzyltrimethylammonium chloride 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 the Toxicity Study Report presents the

experimental results and conclusions fully and clearly.

Mohamed S. Abdel-Rahman, Ph.D.

Department of Pharmacology and Physiology

New Jersey Medical School

Newark, NJ

James S. Bus, Ph.D.

Health and Environmental Sciences

Dow Chemical Company

Midland, MI

Linda A. Chatman, D.V.M.

Pfizer, Inc.

Groton, CT
4

CONTENTS

ABSTRACT ......................................................... 5

INTRODUCTION ...................................................... 7

Chemical and Physical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Production, Use, and Human Exposure .................................... 7

Absorption, Distribution, Metabolism, and Excretion . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Neurotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Carcinogenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Genetic Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Study Rationale and Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Procurement and Characterization of Benzyltrimethylammonium Chloride . . . . . . . . . . . . . . 11

Preparation and Analysis of Dose Formulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

16-Day Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

13-Week Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Statistical Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Quality Assurance Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Genetic Toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Genetic Toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

APPENDIXES

Appendix A Summary of Nonneoplastic Lesions in Rats and Mice . . . . . . . . . . . . . . . . A-1

Appendix B Functional Observation Battery Results . . . . . . . . . . . . . . . . . . . . . . . . . B-1

Appendix C Hematology and Clinical Chemistry Results . . . . . . . . . . . . . . . . . . . . . . C-1

Appendix D Organ Weights and Organ-Weight-to-Body-Weight Ratios . . . . . . . . . . . . . D-1

Appendix E Reproductive Tissue Evaluations and Estrous Cycle Characterization . . . . . . E-1

Appendix F Genetic Toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F-1
5

ABSTRACT

C10H16NCl

BENZYLTRIMETHYLAMMONIUM CHLORIDE

CAS No. 56-93-9

Molecular Weight: 185.70

Synonyms: Ammonium, benzyltrimethyl-chloride (8Cl); BTM; BTMAC; N,N,N,-trimethyl-benzenemethanaminium chloride;

trimethylbenzylammonium chloride (9Cl); TMBAC

Benzyltrimethylammonium chloride is widely used as a solvent for cellulose, a gelling inhibitor in polyester

resins, a chemical intermediate, a paint dispersant, and an acrylic dyeing agent. It is also used in plant growth

regulator compositions and synthetic processes. The National Institute of Environmental Health Sciences

nominated benzyltrimethylammonium chloride for study due to its high production volume and the potential

for occupational exposure, as well as the limited information on toxicity of this chemical. Male and female

F344/N rats and B6C3F1 mice received benzyltrimethylammonium chloride by gavage for 16 days or 13 weeks.

Animals were evaluated for hematology, clinical chemistry, histopathology, neurotoxicity, and reproductive

toxicity. Genetic toxicology studies were conducted in Salmonella typhimurium and in mouse peripheral blood

erythrocytes.

In the 16-day studies, groups of five male and five female rats received 0, 16, 32, 63, 125, or 250 mg

benzyltrimethylammonium chloride/kg body weight in deionized water by gavage, 5 days per week for 16

days. Groups of five male and five female mice received 0, 63, 125, 250, 500, or 1,000 mg/kg

benzyltrimethylammonium chloride in deionized water by gavage, 5 days per week for 16 days. All rats in

the 125 and 250 mg/kg groups, all mice in the 250, 500, and 1,000 mg/kg groups, and one 125 mg/kg female

mouse died on day 1 of the studies. Clinical findings observed in 125 mg/kg male and female rats included

abnormal breathing, ataxia, lethargy (males only), nasal and eye discharge, and tremors. Salivation was

slightly increased in male and female rats in the 63 mg/kg groups. Female mice in the 125 mg/kg group had
6 Benzyltrimethylammonium Chloride, NTP TOX 57

a significantly greater absolute liver weight than that of the vehicle controls. No gross or microscopic changes

observed in rats or mice were considered related to chemical administration.

In the 13-week studies, groups of 10 male and 10 female rats and mice received benzyltrimethylammonium

chloride in deionized water by gavage at doses of 0, 12.5, 25, 50, or 100 mg/kg, 5 days per week for 13

weeks. Benzyltrimethylammonium chloride generally had little effect on the body weights of rats or mice.

Final mean body weights of dosed animals were within 8% (rats) or 3% (mice) of the control group body

weights. The deaths of two female rats and one male and one female mouse administered 100 mg/kg were the

result of pharmacologic effects on the cardiovascular system. Some cholinergic effects including

chromodacryorrhea, lacrimation, salivation, pupillary constriction, altered gait, and mild tremors were

observed at nonlethal doses in rats; these effects were accompanied by alterations in body position. No

significant target organ toxicity was observed in dosed rats or mice.

Benzyltrimethylammonium chloride was not mutagenic in S. typhimurium strain TA97, TA98, TA100, or

TA1535, with or without S9 metabolic activation enzymes. However, significant increases in the frequency

of micronucleated normochromatic erythrocytes were found in the peripheral blood of male and female mice

administered benzyltrimethylammonium chloride by gavage for 13 weeks.

Based on the mortality observed in the 16-day and 13-week studies, rats and mice appeared to be equally

sensitive to benzyltrimethylammonium chloride. The minimally toxic dose for rats and mice was estimated to

be 50 mg/kg.
7

INTRODUCTION

CHEMICAL AND PHYSICAL PROPERTIES

Benzyltrimethylammonium chloride is a quaternary ammonium compound with a structural resemblance to

acetylcholine and other chemicals having cholinergic activity. It is an off-white to yellow powder (SigmaAldrich, 1988) with a melting point of 236E to 239E C (Karsai et al., 1986). It decomposes at 239E C, and

the decomposition products include carbon monoxide, carbon dioxide, nitrogen oxides, hydrochloride gas, and

ammonia (Sigma-Aldrich, 1988; Sax and Lewis, 1989). Benzyltrimethylammonium chloride is hygroscopic

(Sigma-Aldrich, 1988) and soluble in water, ethanol, and butanol and slightly soluble in butyl phthalate and

tributyl phosphate (Sax and Lewis, 1987; Weast, 1989).

PRODUCTION, USE, AND HUMAN EXPOSURE

Benzyltrimethylammonium chloride is prepared by boiling benzyl chloride and trimethylamine in absolute

ethanol. In addition, a reaction involving benzyl dimethylamine and methyl chloride, with or without solvent,

may be used to manufacture this compound (Karsai et al., 1986). Benzyltrimethylammonium chloride is also

prepared by dissolving phenylmethyl chloride in ether and adding 25% trimethylamine in methanol. The

product is collected and recrystallized from alcohol and ether (Hume and Holland, 1965).

In 1983, 16 companies were listed as manufacturers of benzyltrimethylammonium chloride. Ten of the

manufacturers reported a total production volume ranging from 1.5 to 15.5 million pounds (USEPA, 1990).

In 1986 and 1988, respectively, 4,132,000 and 3,985,000 pounds of benzyltrimethylammonium chloride were

produced (USITC, 1986, 1987, 1989). 

Benzyltrimethylammonium chloride is used as a solvent for cellulose, a gelling inhibitor in polyester resins,

a chemical intermediate (Sax and Lewis, 1987), and a paint dispersant for the rubber industry (Chemical

Marketing Reporter, 1983). It is also used extensively as an acrylic dyeing agent in the textile industry (Moore

et al., 1987). Benzyltrimethylammonium chloride is patented for use in plant growth regulator compositions

and synthetic processes (Karsai et al., 1986).
8 Benzyltrimethylammonium Chloride, NTP TOX 57

Occupational exposure may result from the use of this compound in the chemical (Sax and Lewis, 1987),

rubber (Chemical Marketing Reporter, 1983), and textile industries (Moore et al., 1987). Data from the

National Occupational Exposure Survey, conducted by the National Institute for Occupational Safety and

Health during the years 1981 to 1983, estimated that 5,000 workers were potentially exposed to

benzyltrimethylammonium chloride (NIOSH, 1990). No exposure or threshold limit values for

benzyltrimethylammonium chloride have been established.

ABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

Experimental Animals

Male Wistar rats intravenously administered benzyltrimethylammonium iodide eliminated the compound from

plasma in two phases. One phase had a half-life of 3 minutes, and the other had a half-life of 70 minutes.

Benzyltrimethylammonium chloride had an overall half-life of 30 minutes following intravenous administration.

Greater than 90% of the dose was eliminated in the urine; 0.9% was eliminated in the bile. The highest tissue

concentration following intravenous dosing was found in the liver and kidney 10 minutes after injection. No

metabolites were found in the urine, bile, or plasma (Neef et al., 1984).

Absorption from the gastrointestinal tract did not exceed 40% for F344/N rats or 15% for B6C3F1 mice

administered 0.63 mg benzyltrimethylammonium chloride (ring-U-14C)/kg body weight by gavage in water and

did not exceed 10% for rats or mice administered 63 mg/kg dermally for 24 hours. Greater than 90% of the

gavage dose was excreted in the urine and feces within 24 hours of administration. There was no evidence of

metabolism of the parent compound in rats or mice because greater than 90% of the excreted radiolabel was

composed of the parent compound (Sanders et al., 1995).

Humans

No absorption, distribution, metabolism, or excretion studies of benzyltrimethylammonium chloride in humans

were found in a review of the literature.
Benzyltrimethylammonium Chloride, NTP TOX 57 9

TOXICITY

Experimental Animals

An acute median lethal oral dose of 250 mg/kg has been reported for rats of an unspecified strain (Dewitt

et al., 1953). The acute oral lethality of 1,600 mg benzyltrimethylammonium chloride/kg body weight in an

unspecified number of male TAC:SWfBr mice was reported to be 100% (Ellis et al., 1980). In that study,

benzyltrimethylammonium chloride was administered orally as a 2% suspension in 0.5% methylcellulose. The

results of those studies indicate that rats are more sensitive than mice to the lethality of

benzyltrimethylammonium chloride; these results were used to select the doses for the 16-day NTP studies.

A 48-hour median lethal concentration for benzyltrimethylammonium chloride in water fleas (Daphnia pulex)

was determined to be 11.94 ppm in an aquatic static bioassay (Moore et al., 1987). Sanders et al. (1995)

studied the acute oral toxicity of 125, 175, 210, and 250 mg benzyltrimethylammonium chloride/kg body

weight in groups of five male F344/N rats (300 to 350 g). Mortality was observed in the 175, 210, and 250

mg/kg groups. In addition, benzyltrimethylammonium chloride induced muscarinic cholinergic symptoms of

salivation and chromodacryorrhea. These symptoms were relieved by atropine injection, but atropine

administration did not reduce mortality.

Humans

No data were found on the acute toxicity of benzyltrimethylammonium chloride in humans. However,

10 human fatalities resulting from overexposure to aryl/alkyl quaternary ammonium compounds have been

reported. Three of these fatalities involved benzalkonium chloride, and the subjects suffered complete

cardiovascular collapse. A common symptom observed in these fatalities was curariform paralysis.

Intramuscular injection reportedly resulted in liver and kidney necrosis (Gosselin et al., 1984). 

NEUROTOXICITY

The cholinergic activities of benzyltrimethylammonium chloride were studied in isolated muscle preparations

in vitro and through quantitation of saliva collected from the submaxillary duct of cats following intravenous

administration (Hamilton and Rubinstein, 1968). Researchers concluded that benzyltrimethylammonium

chloride is capable of stimulating both nicotinic and muscarinic receptors and speculated that it acts at the same

ganglionic site as acetylcholine. This was confirmed by studies in which 25 mg benzyltrimethylammonium

chloride/kg body weight, dissolved in tetrahydrofurfuryl alcohol and administered intravenously, induced a

gastrocnemius muscle twitch in an unspecified strain of rats (Ellis et al., 1980). Benzyltrimethylammonium

chloride (40 μg/kg), administered without atropine to mongrel dogs via a femoral vein canula, mimicked
10 Benzyltrimethylammonium Chloride, NTP TOX 57

muscarinic acetylcholinergic activity and caused a decrease in blood pressure. A 400 μg/kg dose administered

to atropinized dogs induced a nicotinic increase in blood pressure (Hume and Holland, 1965).

Anticholinergic actions of benzyltrimethylammonium bromide have been demonstrated in vitro using superfused

frog rectus abdominis muscle and rabbit sciatic nerve gastrocnemius muscle preparations (Dretchen et al.,

1971). At low concentrations, the salt inhibited cholinergic receptors. At high concentrations, the salt

appeared to have acted by a nonspecific mechanism that may have involved increased potassium efflux.

CARCINOGENICITY

No carcinogenicity studies of benzyltrimethylammonium chloride in experimental animals or humans were

found in a review of the literature.

GENETIC TOXICITY

Only one set of published mutagenicity data for benzyltrimethylammonium chloride was identified; therefore,

there is insufficient information to fully characterize the genetic activity of this compound. Results of

mutagenicity testing in Salmonella typhimurium strains TA97, TA98, TA100, and TA1535, with and without

induced rat or hamster liver S9 activation enzymes, were uniformly negative (Zeiger et al., 1988).

Concentrations of benzyltrimethylammonium chloride ranged from 100 to 10,000 μg/plate. Slight toxicity was

noted at the higher doses in some trials, but no increases in the number of mutant colonies were seen.

STUDY RATIONALE AND DESIGN

Benzyltrimethylammonium chloride was nominated for toxicity testing by the National Institute of

Environmental Health Sciences because of its high production volume, the potential for occupational exposure,

and the limited information on toxicity. Sixteen-day and 13-week toxicity studies were conducted in

F344/N rats and B6C3F1 mice administered benzyltrimethylammonium chloride by gavage. This route was

selected because benzyltrimethylammonium chloride was found to be poorly absorbed via the dermal route

(Sanders et al., 1995). Micronucleus tests were conducted on mouse peripheral blood erythrocytes following

treatment with benzyltrimethylammonium chloride by gavage for 13 weeks.
11

MATERIALS AND METHODS

PROCUREMENT AND CHARACTERIZATION

OF BENZYLTRIMETHYLAMMONIUM CHLORIDE

Benzyltrimethylammonium chloride was obtained from Fluka Chemical Corporation (Ronkonkoma, NY) in

one lot (306793/1). Information on the identity, purity, and stability of the bulk chemical was provided by the

manufacturer; identity was confirmed by the study laboratory. Reports on analyses performed in support of

the benzyltrimethylammonium chloride studies are on file at the National Institute of Environmental Health

Sciences.

The manufacturer identified the chemical, an off-white to yellow crystalline powder, as

benzyltrimethylammonium chloride by nuclear magnetic resonance spectroscopy. The purity of lot 306793/1,

determined by argentometric titration, was 100.4% or greater. The study laboratory confirmed the identity

of the chemical with infrared spectroscopy. The spectrum was consistent with a literature reference for

benzyltrimethylammonium bromide (Aldrich, 1990).

Based on the manufacturerís stability information, the bulk chemical was stored at room temperature in sealed

containers flushed with nitrogen to expel moisture.

PREPARATION AND ANALYSIS OF DOSE FORMULATIONS

The dose formulations for the 16-day studies were prepared once within 7 days of the beginning of dosing.

Dose formulations for the 13-week studies were prepared within 8 days of the first day of dosing, then every

2 to 3 weeks until the end of the study. To prepare the dose formulations, a weighed amount of

benzyltrimethylammonium chloride was dissolved in deionized water and diluted to achieve the highest

concentration required. Serial dilutions of the highest concentration were made to obtain each of the lower

concentrations. Dose formulations were stored in sealed serum vials (16-day studies) or amber glass bottles

(13-week studies) at room temperature.
12 Benzyltrimethylammonium Chloride, NTP TOX 57

Stability studies of 3.2 and 1.25 mg/mL dose formulations were performed by the study laboratory using

ultraviolet spectroscopy. Stability was confirmed for at least 21 days (3.2 mg/mL) or 28 days (1.25 mg/mL)

for formulations stored in sealed containers at room temperature.

The study laboratory analyzed the dose formulations used in the 16-day studies and the initial, mid-point, and

final dose formulations used in the 13-week studies. The study laboratory also analyzed animal room samples

of the same dose formulations after they had been in use for 2 to 3 weeks. All dose formulations administered

to rats and mice and all animal room samples were within 10% of the target concentrations.

16-DAY STUDIES

Male and female F344/N rats and B6C3F1 mice were obtained from Taconic Farms (Germantown, NY). On

receipt, the rats and mice were 4 weeks old. Animals were quarantined for 13 (rats) or 14 (mice) days and

were 6 weeks old on the first day of the studies. Groups of five male and five female rats received 0, 16, 32,

63, 125, or 250 mg benzyltrimethylammonium chloride per kg body weight in deionized water by gavage,

5 days per week for 16 days. Groups of five male and five female mice received 0, 63, 125, 250, 500, or

1,000 mg/kg in deionized water by gavage, 5 days per week for 16 days. Feed and water were available

ad libitum. Rats and female mice were housed five per cage; male mice were housed individually. Animals

were observed daily, and clinical findings and body weights were recorded initially, on day 8, and at the end

of the studies. Prior to terminal sacrifice, a functional observation battery was performed on all surviving rats.

Details of the study design and animal maintenance are summarized in Table 1.

At the end of the 16-day studies, the animals were anesthetized with a mixture of carbon dioxide and oxygen,

and blood was collected from the retroorbital sinus of all rats and mice for hematology and clinical chemistry

analyses. Blood samples for hematology analysis were collected in tubes containing EDTA as an anticoagulant.

Blood samples for clinical chemistry analysis were collected in untreated clot tubes, and the sera were separated

by centrifugation. Hematology parameters were analyzed on a Serono-Baker 9000 Automated Cell Counter

(Serono-Baker Diagnostics, Allentown, PA). Manual hematocrit values were determined using an Adams

Microhematocrit Centrifuge, CT2900 (Clay Adams, Sparks, MD). Leukocyte differentials and erythrocyte,

leukocyte, lymphocyte, and platelet morphology were determined by light microscopy from blood smears

stained with modified Wrightís stain using an Ames Hematek Slide Stainer (Miles Laboratory, Ames Division,

Elkhart, IN). Reticulocyte counts were determined by light microscopy using blood samples stained with new

methylene blue (Sigma Chemical Company, St. Louis, MO). Clinical chemistry parameters were measured
Benzyltrimethylammonium Chloride, NTP TOX 57 13

using a Hitachi 717Æ chemistry analyzer (Boehringer Mannheim, Indianapolis, IN). All reagents were obtained

from the manufacturer with the exception of the reagents for sorbitol dehydrogenase and total bile acid

determinations, which were obtained from Sigma Chemical Company. The parameters measured are listed

in Table 1.

A necropsy was performed on all rats and mice. The heart, right kidney, liver, lung, spleen, right testis, and

thymus were weighed. Tissues for microscopic examination were fixed and preserved in 10% neutral buffered

formalin, processed and trimmed, embedded in paraffin, sectioned to a thickness of 5 μm, and stained with

hematoxylin and eosin. Histopathologic examinations were performed on vehicle control rats and mice, rats

receiving 63, 125, or 250 mg/kg, and mice receiving 125, 250, 500, or 1,000 mg/kg. Table 1 lists the tissues

and organs examined.

13-WEEK STUDIES

Male and female F344/N rats and B6C3F1 mice were obtained from Taconic Farms (Germantown, NY). Upon

receipt, the rats and mice were 4 weeks old. Animals were quarantined for 12 to 15 days and were 6 weeks

old on the first day of the studies. Before the studies began, five male and five female rats and mice were

randomly selected for parasite evaluation and gross observation for evidence of disease. Blood was collected

from five male and five female control rats and untreated mice at the end of the 13-week studies. The sera

were analyzed for antibody titers to rodent viruses (Boorman et al., 1986; Rao et al., 1989a,b). All results

were negative.

Core study groups of 10 male and 10 female rats and mice received benzyltrimethylammonium chloride in

deionized water by gavage at doses of 0, 12.5, 25, 50, or 100 mg/kg, 5 days per week for 13 weeks. Feed

and water were available ad libitum. Rats and female mice were housed five per cage; male mice were housed

individually. Clinical findings were recorded and animals were weighed initially, on day 8, and weekly until

the end of the studies. A functional observation battery was performed on core study rats on days 10 and 85.

Details of the study design and animal maintenance are summarized in Table 1.

On days 3 and 21, blood was collected from the retroorbital sinus of groups of 10 special study rats

administered the same doses as core study rats for hematology and clinical chemistry analyses. At the end of

the 13-week studies, blood was collected from the retroorbital sinus of all core study rats for hematology and

clinical chemistry analyses and from all core study mice for clinical chemistry analyses. Methods used for
14 Benzyltrimethylammonium Chloride, NTP TOX 57

hematology and clinical chemistry analyses were the same as those used in the 16-day studies. The parameters

measured are listed in Table 1.

At the end of the 13-week studies, samples were collected for sperm motility and vaginal cytology evaluations

on core study rats and mice receiving 0, 25, 50, or 100 mg/kg. The parameters evaluated are listed in Table 1.

Methods used were those described in the NTPís sperm morphology and vaginal cytology evaluations protocol

(NTP, 1991). For 12 consecutive days prior to the scheduled terminal sacrifice, the vaginal vaults of the

females were moistened with saline, if necessary, and samples of vaginal fluid and cells were stained. Relative

numbers of leukocytes, nucleated epithelial cells, and large squamous epithelial cells were determined and used

to ascertain estrous cycle stage (i.e., diestrus, proestrus, estrus, and metestrus). Male animals were evaluated

for sperm count and motility. The left testis and left epididymis were isolated and weighed. The tail of the

epididymis (cauda epididymis) was then removed from the epididymal body (corpus epididymis) and weighed.

Test yolk (rats) or modified Tyrodeís buffer (mice) was applied to slides and a small incision was made at the

distal border of the cauda epididymis. The sperm effluxing from the incision were dispersed in the buffer on

the slides, and the numbers of motile and nonmotile spermatozoa were counted for five fields per slide by

two observers. Following completion of sperm motility estimates, each left cauda epididymis was placed in

buffered saline solution. Caudae were minced, and the tissue was incubated in the saline solution and then heat

fixed at 65E C. Sperm density was then determined microscopically with the aid of a hemacytometer. To

quantify spermatogenesis, the testicular spermatid head count was determined by removing the tunica albuginea

and homogenizing the left testis in phosphate-buffered saline containing 10% dimethyl sulfoxide.

Homogenization-resistant spermatid nuclei were counted with a hemacytometer.

A necropsy was performed on all core study rats and mice. The heart, right kidney, liver, lung, right testis,

and thymus were weighed. Tissues for microscopic examination were fixed and preserved in 10% neutral

buffered formalin, processed and trimmed, embedded in paraffin, sectioned to a thickness of 5 μm, and stained

with hematoxylin and eosin. A complete histopathologic examination was performed on vehicle control groups

and all 100 mg/kg rats and mice. Table 1 lists the tissues and organs routinely examined.

Upon completion of the laboratory pathologistís histopathologic evaluation, the slides, paraffin blocks, and

residual wet tissues were sent to the NTP Archives for inventory, slide/block match, and wet tissue audit. The

slides, individual animal data records, and pathology tables were sent to an independent pathology laboratory

where quality assessment was performed. Results were reviewed and evaluated by the NTP. Details of these

review procedures have been described by Maronpot and Boorman (1982) and Boorman et al. (1985).
Benzyltrimethylammonium Chloride, NTP TOX 57 15

TABLE 1

Experimental Design and Materials and Methods in the Gavage Studies 

of Benzyltrimethylammonium Chloride

16-Day Studies 13-Week Studies

Study Laboratory

Microbiological Associates, Inc. (Bethesda, MD) Microbiological Associates, Inc. (Bethesda, MD)

Strain and Species

Rats: F344/N

Mice: B6C3F1

Rats: F344/N

Mice: B6C3F1

Animal Source

Taconic Farms 

(Germantown, NY)

Taconic Farms

(Germantown, NY)

Time Held Before Studies

Rats: 13 days

Mice: 14 days

Rats: 12 days (males) or 13 days (females)

Mice: 14 days (males) or 15 days (females)

Average Age When Studies Began

6 weeks 6 weeks

Date of First Dose

Rats: 10 August 1992

Mice: 11 August 1992

Rats: 12 (males) or 13 (females) October 1993

Mice: 14 (males) or 15 (females) October 1993

Duration of Dosing

16 days (5 days/week) 13 weeks (5 days/week)

Date of Last Dose

Rats: 25 August 1992

Mice: 26 August 1992

Rats: 10 (males) or 11 (females) January 1994

Mice: 12 (males) or 13 (females) January 1994

Necropsy Dates

Rats: 26 August 1992

Mice: 27 August 1992

Rats: 11 (males) or 12 (females) January 1994

Mice: 13 (males) or 14 (females) January 1994

Average Age at Necropsy

8 weeks 19 weeks

Size of Study Groups

5 males and 5 females 10 males and 10 females

Method of Distribution

Animals were distributed randomly into groups of approximately

equal initial mean body weights.

Same as 16-day studies

Animals per cage

Rats: 5

Mice: 1 (males) or 5 (females)

Rats: 5

Mice: 1 (males) or 5 (females)

Method of Animal Identification

Tail tattoo Tail tattoo

Diet

NIH-07 open formula pelleted diet (Zeigler Brothers, Inc.,

Gardners, PA), available ad libitum, changed weekly

NIH-07 open formula meal diet (Zeigler Brothers, Inc., 

Gardners, PA), available ad libitum, changed weekly
16 Benzyltrimethylammonium Chloride, NTP TOX 57

TABLE 1

Experimental Design and Materials and Methods in the Gavage Studies 

of Benzyltrimethylammonium Chloride

16-Day Studies 13-Week Studies

Water

Tap water (Washington Suburban Sanitary Commission 

Potomac Plant) via automatic watering system (Edstrom

Industries, Inc., Waterford, WI), available ad libitum

Same as 16-day studies

Cages

Polycarbonate (Lab Products, Inc., Rochelle Park, NJ), changed

twice weekly for group-housed animals and once weekly for

individually housed animals

Same as 16-day studies

Bedding

Sani-ChipsÆ (P.J. Murphy Forest Products, Montville, NJ)

changed twice weekly for group-housed animals and once weekly

for individually housed animals 

Same as 16-day studies

Racks

Stainless steel (Lab Products, Inc., Rochelle Park, NJ), changed

and rotated every 2 weeks

Same as 16-day studies

Animal Room Environment

Temperature: 20.6E-23.9E C

Relative humidity: 35%-65%

Room fluorescent light: 12 hours/day

Room air changes: 10/hour

Temperature: 20.6E-23.9E C

Relative humidity: 35%-65%

Room fluorescent light: 12 hours/day

Room air changes: 10/hour

Doses

Rats: 0, 16, 32, 63, 125, or 250 mg/kg in deionized water by

gavage (dosing volume=5 mL/kg body weight)

Mice: 0, 63, 125, 250, 500, or 1,000 mg/kg in deionized water

by gavage (dosing volume=10 mL/kg body weight)

0, 12.5, 25, 50, or 100 mg/kg in deionized water by gavage

(dosing volume=5 mL/kg body weight for rats and 10 mL/kg 

body weight for mice)

Type and Frequency of Observation

Observed twice daily; clinical findings and body weights were

recorded initially, on day 8, and at the end of the studies.

Observed twice daily; animals were weighed and clinical findings

were recorded initially, on day 8, and weekly thereafter until the

end of the studies.

Method of Sacrifice

CO2 asphyxiation Same as 16-day studies

Necropsy

Necropsy performed on all animals. Organs weighed were the

heart, right kidney, liver, lung, spleen, right testis, and thymus.

Necropsy performed on all core study animals. Organs weighed

were the heart, right kidney, liver, lung, right testis, and thymus.
Benzyltrimethylammonium Chloride, NTP TOX 57 17

TABLE 1

Experimental Design and Materials and Methods in the Gavage Studies 

of Benzyltrimethylammonium Chloride

16-Day Studies 13-Week Studies

Clinical Pathology

Blood was collected from the retroorbital sinus of all rats and

mice surviving to the end of the studies for hematology and

clinical chemistry.

Hematology: automated and manual hematocrit; hemoglobin

concentration; erythrocyte, reticulocyte, and nucleated

erythrocyte counts; mean cell volume; mean cell hemoglobin;

mean cell hemoglobin concentration; platelet count; and total

leukocyte count and differentials

Clinical chemistry: urea nitrogen, creatinine, total protein,

albumin, alanine aminotransferase, alkaline phosphatase, creatine

kinase, sorbitol dehydrogenase, serum cholinesterase, erythrocyte

cholinesterase, and bile acids

Blood was collected from the retroorbital sinus of all special study

rats on days 3 and 21 and from all core study rats surviving until

the end of the study for hematology and clinical chemistry. 

Blood was collected from the retroorbital sinus of all mice

surviving to the end of the study for clinical chemistry.

Hematology: automated and manual hematocrit; hemoglobin

concentration; erythrocyte, reticulocyte, and nucleated

erythrocyte counts; mean cell volume; mean cell hemoglobin;

mean cell hemoglobin concentration; platelet count; and total

leukocyte count and differentials

Clinical chemistry: urea nitrogen, creatinine, total protein,

albumin, alanine aminotransferase, alkaline phosphatase, creatine

kinase, sorbitol dehydrogenase, serum cholinesterase, and bile

acids

Histopathology

Complete histopathology was performed on all vehicle control

animals, 63, 125, and 250 mg/kg rats, and 125, 250, 500, and

1,000 mg/kg mice. In addition to gross lesions and tissue

masses, the following tissues were examined: adrenal gland,

bone and marrow, brain, clitoral gland, esophagus, gallbladder

(mice), heart, large intestine (cecum, colon, rectum), small

intestine (duodenum, jejunum, ileum), kidney, liver, lung, lymph

nodes (mandibular and mesenteric), mammary gland (except male

mice), nose, ovary, pancreas, parathyroid gland, pituitary gland,

preputial gland, prostate gland, salivary gland, skin, spleen,

stomach (forestomach and glandular), testis (with epididymis and

seminal vesicle), thymus, thyroid gland, trachea, urinary bladder,

uterus, and Zymbalís gland (rats).

Complete histopathology was performed on all core study vehicle

control and 100 mg/kg rats and mice. In addition to gross lesions

and tissue masses, the following tissues were examined: adrenal

gland, bone with marrow, brain, clitoral gland, esophagus,

gallbladder (mice), heart, large intestine (cecum, colon, rectum),

small intestine (duodenum, jejunum, ileum), kidney, liver, lung,

lymph nodes (mandibular and mesenteric), mammary gland,

muscle, nose, ovary, pancreas, parathyroid gland, pituitary

gland, preputial gland, prostate gland, salivary gland, spinal cord,

spleen, stomach (forestomach and glandular), testis (with

epididymis and seminal vesicle), thymus, thyroid gland, trachea,

urinary bladder, uterus, and Zymbalís gland (rats). The lung of

all 50 mg/kg rats was also examined.

Sperm Motility and Vaginal Cytology

None At the end of the studies, sperm samples were collected from all

core study male rats and mice in the 0, 25, 50, and 100 mg/kg

groups for sperm motility evaluations. The following parameters

were evaluated: spermatid heads per testis and per gram testis,

spermatid counts, and epididymal spermatozoal motility and

concentration. The left cauda epididymis, left epididymis, and

left testis were weighed. Vaginal samples were collected for up

to 12 consecutive days prior to the end of the studies from all

core study females administered 0, 25, 50, or 100 mg/kg for

vaginal cytology evaluations. The parameters evaluated were

estrous cycle length and the percentage of cycle spent in the

estrous cycle stages.

Functional Observation Battery

At the end of the study, all surviving rats were subjected to a

functional observation battery. The parameters evaluated were 

body position, activity level, coordination, gait, general behavior,

head-flick, head-searching, compulsive licking or biting,

backward walking, self-mutilation, circling, convulsions,

tremors, lacrimation or chromodacryorrhea, salivation,

piloerection, pupillary dilation or constriction, unusual

respiration, diarrhea, excessive or diminished urination, and

vocalization.

Core study rats were subjected to a functional observation battery

on days 10 and 85. The parameters evaluated were the same as

those evaluated in the 16-day studies.
18 Benzyltrimethylammonium Chloride, NTP TOX 57

STATISTICAL METHODS

Calculation and Analysis of Lesion Incidences

The incidences of lesions presented in Appendix A are given as the number of animals bearing such lesions

at a specific anatomic site and the number of animals with that site examined microscopically. The Fisher

exact test, a procedure based on the overall proportion of affected animals, was used to determine significance

(Gart et al., 1979).

Analysis of Continuous Variables

Two approaches were employed to assess the significance of pairwise comparisons between dosed and vehicle

control groups in the analysis of continuous variables. Organ and body weight data, which have approximately

normal distributions, were analyzed with the parametric multiple comparison procedures of Dunnett (1955) and

Williams (1971, 1972). Hematology, clinical chemistry, spermatid, and epididymal spermatozoal data, which

have typically skewed distributions, were analyzed using the nonparametric multiple comparison methods of

Shirley (1977) and Dunn (1964). Jonckheereís test (Jonckheere, 1954) was used to assess the significance of

the dose-related trends and to determine whether a trend-sensitive test (Williamsí or Shirleyís test) was more

appropriate for pairwise comparisons than a test that does not assume a monotonic dose-related trend (Dunnettís

or Dunnís test). Prior to statistical analysis, extreme values identified by the outlier test of Dixon and Massey

(1951) were examined by NTP personnel, and implausible values were eliminated from the analysis. Because

vaginal cytology data are proportions (the proportion of the observation period that an animal was in a given

estrous stage), an arcsine transformation was used to bring the data into closer conformance with a normality

assumption. Treatment effects were investigated by applying a multivariate analysis of variance (Morrison,

1976) to the transformed data to data to test for simultaneous equality of measurements across doses. The

Fisher exact test was used to determine the significance of the functional observation battery data (Gart et al.,

1979).

QUALITY ASSURANCE METHODS

The 13-week studies of benzyltrimethylammonium chloride were conducted in compliance with Food and Drug

Administration Good Laboratory Practice Regulations (21 CFR, Part 58). The quality assurance unit of

Microbiological Associates, Inc., performed audits and inspections of protocols, procedures, data, and reports

throughout the course of the studies.
Benzyltrimethylammonium Chloride, NTP TOX 57 19

GENETIC TOXICOLOGY

Salmonella typhimurium Mutagenicity Test Protocol

Testing was performed as reported by Zeiger et al. (1988). Benzyltrimethylammonium chloride was sent to

the laboratory as a coded aliquot from Radian Corporation (Austin, TX). It was incubated with the Salmonella

typhimurium tester strains TA97, TA98, TA100, and TA1535, either in buffer or S9 mix (metabolic activation

enzymes and cofactors from Aroclor 1254-induced male Sprague-Dawley rat or Syrian hamster liver) for

20 minutes at 37E C. Top agar supplemented with L-histidine and d-biotin was added, and the contents of the

tubes were mixed and poured onto the surfaces of minimal glucose agar plates. Histidine-independent mutant

colonies arising on these plates were counted following incubation for 2 days at 37E C.

Each trial consisted of triplicate plates of concurrent positive and negative controls and five doses of

benzyltrimethylammonium chloride. In the absence of toxicity, 10,000 μg/plate was selected as the high dose.

In this assay, a positive response is defined as a reproducible, dose-related increase in histidine-independent

(revertant) colonies in any one strain/activation combination. An equivocal response is defined as an increase

in revertants that is not dose related, is not reproducible, or is not of sufficient magnitude to support a

determination of mutagenicity. A negative response is obtained when no increase in revertant colonies is

observed following chemical treatment. There is no minimum percentage or fold increase required for a

chemical to be judged positive or weakly positive.

Mouse Peripheral Blood Micronucleus Test Protocol

A detailed discussion of this assay is presented by MacGregor et al. (1990). At the end of the 13-week toxicity

study, peripheral blood samples were obtained from male and female mice. Smears were immediately prepared

and fixed in absolute methanol. The methanol-fixed slides were stained with acridine orange and coded. Slides

were scanned to determine the frequency of micronuclei in 1,000 normochromatic erythrocytes (NCEs) in up

to 10 animals per dose group.

The results were tabulated as the mean of the pooled results from all animals within a treatment group plus or

minus the standard error of the mean. The frequency of micronucleated cells among NCEs was analyzed by

a statistical software package that tested for increasing trend over dose groups with a one-tailed CochranArmitage trend test, followed by pairwise comparisons between each dose group and the control group (ILS,

1990). In the presence of excess binomial variation, as detected by a binomial dispersion test, the binomial

variance of the Cochran-Armitage test was adjusted upward in proportion to the excess variation. In the
20 Benzyltrimethylammonium Chloride, NTP TOX 57

micronucleus test, an individual trial is considered positive if the trend test P value is less than or equal to 0.025

or if the P value for any single dose group is less than or equal to 0.025 divided by the number of dose groups.

A final call of positive for micronucleus induction is preferably based on reproducibly positive trials (as noted

above). Results of the 13-week studies were accepted without repeat tests, because additional test data could

not be obtained. Ultimately, the final call is determined by the scientific staff after considering the results of

statistical analyses, the reproducibility of any effects observed, and the magnitudes of those effects.

Evaluation Protocol

These are the basic guidelines for arriving at an overall assay result for assays performed by the National

Toxicology Program. Statistical as well as biological factors are considered. For an individual assay, the

statistical procedures for data analysis have been described in the preceding protocols. There have been

instances, however, in which multiple aliquots of a chemical were tested in the same assay, and differing

results were obtained among aliquots and/or among laboratories. Results from more than one aliquot or from

more than one laboratory are not simply combined into an overall result. Rather, all the data are critically

evaluated, particularly with regard to pertinent protocol variations, in determining the weight of evidence for

an overall conclusion of chemical activity in an assay. In addition to multiple aliquots, the in vitro assays have

another variable that must be considered in arriving at an overall test result. In vitro assays are conducted with

and without exogenous metabolic activation. Results obtained in the absence of activation are not combined

with results obtained in the presence of activation; each testing condition is evaluated separately. The results

presented in the Abstract of this Toxicity Study Report represent a scientific judgement of the overall evidence

for activity of the chemical in an assay.
21

RESULTS

RATS

16-DAY STUDY

All male and female rats in the 125 and 250 mg/kg groups died on day 1 of the study (Table 2). All other rats

survived to the end of the study. The final mean body weights and body weight gains of dosed males and

females were similar to those of the vehicle controls (Table 2).

TABLE 2

Survival and Body Weights of Rats in the 16-Day Gavage Study of Benzyltrimethylammonium Chloride

Mean Body Weightb (g) Final Weight

Dose Survivala Initial Final Change Relative to Controls

(mg/kg) (%)

Male

0 5/5 151 ± 3 240 ± 4 89 ± 2 

16 5/5 150 ± 3 231 ± 5 81 ± 4 96

32 5/5 147 ± 7 233 ± 12 86 ± 6 97

63 5/5 146 ± 4 226 ± 8 80 ± 5 94

125 0/5c 150 ± 4 ó ó ó

250 0/5c 149 ± 5 ó ó ó

Female

0 5/5 103 ± 4 137 ± 5 34 ± 1

16 5/5 103 ± 3 144 ± 5 41 ± 2 105

32 5/5 106 ± 2 142 ± 4 36 ± 2 104

63 5/5 103 ± 2 137 ± 4 34 ± 2 100

125 0/5c 103 ± 2 ó ó ó

250 0/5c 105 ± 4 ó ó ó

a Number of animals surviving at 16 days/number initially in group b Weights and weight changes are given as mean ± standard error. Differences from the vehicle control groups were not significant by

Dunnettís test. No data were calculated for groups with 100% mortality. c Day of death: 1
22 Benzyltrimethylammonium Chloride, NTP TOX 57

Chemical-related clinical findings observed in three males and one female administered 125 mg/kg included

abnormal breathing, ataxia, lethargy (males only), nasal and eye discharge, and tremors on day 1; one

63 mg/kg male was also lethargic and had nasal and eye discharge. A functional observation battery was

performed on males and females surviving to the end of the study. All males in the 63 mg/kg group exhibited

salivation (Table B1). Compared to the vehicle controls, incidences of pupillary dilation and mild tremors were

slightly, but not significantly, increased in 63 mg/kg males. The only chemical-related effect observed in

females was salivation in two of five rats in the 63 mg/kg group.

Because of 100% mortality in the 125 and 250 mg/kg groups, no hematology or clinical chemistry evaluations

were performed and no organ weight data were collected for these groups. For the groups with survivors,

there were no treatment-related changes in the hematology, clinical chemistry, or organ weight variables

(Tables C1 and D1). No chemical-related gross or microscopic changes were observed. In some animals that

died early, necrosis of thymic lymphocytes and pulmonary edema were observed; these were thought to be

stress-related or agonal changes. Based on the 100% mortality observed at 125 and 250 mg/kg, doses of 0,

12.5, 25, 50, and 100 mg/kg were selected for the 13-week gavage study in rats.
Benzyltrimethylammonium Chloride, NTP TOX 57 23

13-WEEK STUDY

One 25 mg/kg and two 100 mg/kg female rats died before the end of the study (Table 3); the deaths of the

100 mg/kg females were considered to be due to pharmacologic effects of benzyltrimethylammonium chloride

on the cardiovascular system. All other rats survived to the end of the study. The mean body weight gain of

100 mg/kg males was significantly less than that of the vehicle controls (Table 3 and Figure 1). Chemicalrelated clinical findings included nasal and eye discharge in 12.5 (1/10), 25 (6/10), 50 (6/10), and

100 (10/10) mg/kg males and in 50 (6/10) and 100 (6/10) mg/kg females, oral discharge in 50 (2/10) and

100 (3/10) mg/kg males and in 100 mg/kg females (9/10), and tremors in 100 mg/kg males (4/10) and

females (2/10). 

TABLE 3

Survival and Body Weights of Rats in the 13-Week Gavage Study of Benzyltrimethylammonium Chloride

Mean Body Weightb (g) Final Weight

Dose Survivala Initial Final Change Relative to Controls

(mg/kg) (%)

Male

0 10/10 125 ± 4 338 ± 8 213 ± 7

12.5 10/10 124 ± 4 337 ± 8 213 ± 6 100

25 10/10 125 ± 3 336 ± 8 211 ± 5 99

50 10/10 125 ± 4 340 ± 5 215 ± 4 101

100 10/10 125 ± 3 311 ± 9 186 ± 7** 92

Female

0 10/10 106 ± 3 190 ± 3 85 ± 2 

12.5 10/10 106 ± 3 198 ± 4 93 ± 2 104

25 9/10c 104 ± 2 193 ± 3 88 ± 2 101

50 10/10 107 ± 3 192 ± 4 85 ± 3 101

100 8/10d 107 ± 3 187 ± 4 81 ± 2 98

** Significantly different (P#0.01) from the vehicle control group by Dunnettís test a Number of animals surviving at 13 weeks/number initially in group b Weights and weight changes are given as mean ± standard error. Subsequent calculations are based on animals surviving to the end of the

study. c Week of death: 12 (gavage accident) d Week of death: 10, 12
24 Benzyltrimethylammonium Chloride, NTP TOX 57

FIGURE 1 

Body Weights of Rats Administered Benzyltrimethylammonium Chloride 

by Gavage for 13 Weeks
Benzyltrimethylammonium Chloride, NTP TOX 57 25

A functional observation battery was conducted on days 10 and 85. Clinical evaluation demonstrated

chromodacryorrhea and increased salivation in male and female rats in the 100 mg/kg group on day 85

(Tables 4 and B2). In female rats, slight lacrimation was observed in all dosed groups (30% to 75%) on

day 85. Chemical-related effects on the motor system were evident on day 85 in male and female rats in the

100 mg/kg groups. These effects were characterized by an altered gait (males: 40%; females: 25%) and

mild to severe tremors (males: 50%; females: 63%) and were accompanied by alterations in motor

coordination and, in some cases, altered body position (males: 40%; females: 38%). Pupillary constriction

was observed in 3 of 10 females in the 50 mg/kg group and 5 of 10 females in the 100 mg/kg group.

TABLE 4

Summary of Functional Observation Battery for Rats on Day 85 in the 13-Week Gavage Study

of Benzyltrimethylammonium Chloride

Vehicle

Control 12.5 mg/kg 25 mg/kg 50 mg/kg 100 mg/kg

Male

n 10 10 10 10 10

Body position

Crouched over 0 0 0 0 2

Head bobbing 00002

Coordination of movement

Moderately impaired 0 0 0 0 1

Severely impaired 0 0 0 0 1

Gait

Hunched or crouched 0 0 0 0 2

Body drags/is flattened 0 0 0 0 2

Lacrimation or chromodacryorrhea

Slight 0 0 0 0 5*

Severe 0 0 0 0 1

Pupillary constriction or dilation

Constricted 0 0 0 0 1

Salivation

Slight 0 1 2 4* 2

Severe 0 0 0 2 8**

Tremors

Mild 0 0 0 0 2

Severe whole body 0000 3
26 Benzyltrimethylammonium Chloride, NTP TOX 57

TABLE 4

Summary of Functional Observation Battery for Rats on Day 85 in the 13-Week Gavage Study

of Benzyltrimethylammonium Chloride

Vehicle

Control 12.5 mg/kg 25 mg/kg 50 mg/kg 100 mg/kg

Female

n 10 10 9 10 8

Body position

Flattened 0 0 0 0 1

Crouched over 0 0 0 0 2

Coordination of movement

Slightly impaired 0 0 0 0 3

Gait

Ataxia 0 0 0 0 2

Lacrimation or chromodacryorrhea

Slight 0 3 3 4* 6**

Pupillary constriction or dilation

Constricted 0 0 0 3 4*

Salivation

Slight 1 1 1 0 3

Severe 0 0 0 1 1

Tremors

Mild 0 0 0 0 4*

Mild whole body 00001

* Significantly different (P#0.05) from the vehicle control group by the Fisher exact test

** P#0.01

Significant differences were observed in the hematology and clinical chemistry variables (Table C2). The

majority of these differences were sporadic or minimal, did not demonstrate a treatment relationship, or were

inconsistent between genders and consequently were not considered to be toxicologically relevant. However,

at week 13, there were very minimal, treatment-related increases in the mean cell volumes of rats. These

increases in mean cell volume, which is an estimate of the average size (expressed as a volume) of a population

of erythrocytes, suggest that the erythrocytes were minimally larger in the dosed animals than in the vehicle

controls. Additionally, females administered 25 mg/kg or greater appeared to have minimally decreased total

protein and albumin concentrations. The biologic significance of the differences in mean cell volumes and

protein concentrations is unknown; because these changes were minimal and no other hematologic, clinical

chemistry, and pathologic alterations occurred, the differences were not considered to be clinically significant.
Benzyltrimethylammonium Chloride, NTP TOX 57 27

Benzyltrimethylammonium chloride administration had no effect on the absolute or relative organ weights of

males or females (Table D2). No chemical-related gross or microscopic lesions were observed (Tables A1 and

A2). There were no differences in reproductive tissue parameters in males (Table E1). A minimal shortening

of diestrus and prolongation of proestrus occurred in 25 mg/kg females; there was no alteration in the length

of the estrous cycle (Table E2).
28 Benzyltrimethylammonium Chloride, NTP TOX 57

MICE

16-DAY STUDY

All male and female mice in the 250, 500, and 1,000 mg/kg groups and one 125 mg/kg female died on day 1

of the study; all other mice survived to the end of the study (Table 5). The mean body weight gains of females

in the 63 and 125 mg/kg groups were significantly greater than that of the vehicle controls (Table 5). The final

mean body weights of dosed males and females and mean body weight gains of dosed males were similar to

those of the vehicle controls. Clinical findings occurred sporadically and were not considered to be related to

chemical administration.

TABLE 5

Survival and Body Weights of Mice in the 16-Day Gavage Study of Benzyltrimethylammonium Chloride

Mean Body Weightb (g) Final Weight

Dose Survivala Initial Final Change Relative to Controls

(mg/kg) (%)

Male

0 5/5 23.8 ± 0.5 26.4 ± 0.7 2.6 ± 0.3

63 5/5 23.8 ± 0.6 26.0 ± 0.5 2.2 ± 0.2 98

125 5/5 23.6 ± 0.4 26.1 ± 0.6 2.6 ± 0.4 99

250 0/5c 23.7 ± 0.4 ó ó ó

500 0/5c 23.5 ± 0.5 ó ó ó

1,000 0/5c 23.7 ± 0.6 ó ó ó

Female

0 5/5 20.0 ± 0.4 21.2 ± 0.4 1.2 ± 0.1

63 5/5 19.2 ± 0.5 21.9 ± 0.5 2.7 ± 0.2** 103

125 4/5c 19.3 ± 0.5 22.4 ± 0.1 2.6 ± 0.3** 105

250 0/5c 19.6 ± 0.3 ó ó ó

500 0/5c 19.7 ± 0.3 ó ó ó

1,000 0/5c 19.4 ± 0.5 ó ó ó

** Significantly different (P#0.01) from the vehicle control group by Dunnettís test a Number of animals surviving at 16 days/number initially in group b Weights and weight changes are given as mean ± standard error. Subsequent calculations are based on animals surviving to the end of the

study. No data were calculated for groups with 100% mortality. c Day of death: 1
Benzyltrimethylammonium Chloride, NTP TOX 57 29

Because of 100% mortality in the groups receiving 250 mg/kg or greater, no hematology or clinical chemistry

evaluations were performed and no organ weight data were collected for these groups. For the groups with

survivors, there were no treatment-related changes in hematology or clinical chemistry variables (Table C3).

For 125 mg/kg females, the absolute liver weight was significantly greater and the relative lung weight was

significantly less than those of the vehicle controls (Table D3). No chemical-related gross or microscopic

changes were observed. Pulmonary congestion and edema were observed in some animals that died early and

were interpreted to be an agonal change. Based on the 100% mortality observed at 250 mg/kg and greater,

doses of 0, 12.5, 25, 50, and 100 mg/kg were selected for the 13-week gavage study in mice.
30 Benzyltrimethylammonium Chloride, NTP TOX 57

13-WEEK STUDY

One male and one female in the 100 mg/kg groups died before the end of the study; the deaths were the result

of pharmacologic effects of benzyltrimethylammonium chloride on the cardiovascular system (Table 6). All

other mice survived until the end of the study. Final mean body weights and body weight gains of dosed males

and females were similar to those of the vehicle controls (Table 6 and Figure 2). Beginning at week 10,

hyperactivity was observed in 100 mg/kg females immediately following administration of

benzyltrimethylammonium chloride. However, the hyperactivity diminished within an hour after dosing. No

other clinical findings were observed.

TABLE 6

Survival and Body Weights of Mice in the 13-Week Gavage Study of Benzyltrimethylammonium Chloride

Mean Body Weightb (g) Final Weight

Dose Survivala Initial Final Change Relative to Controls

(mg/kg) (%)

Male

0 10/10 24.4 ± 0.5 34.9 ± 0.7 10.6 ± 0.5 

12.5 10/10 24.3 ± 0.5 34.9 ± 1.0 10.6 ± 0.5 100

25 10/10 24.3 ± 0.5 34.5 ± 0.6 10.1 ± 0.5 99

50 10/10 24.6 ± 0.5 34.8 ± 0.9 10.2 ± 0.5 100

100 9/10c 24.4 ± 0.5 33.9 ± 0.9 9.4 ± 0.4 97

Female

0 10/10 19.3 ± 0.4 29.1 ± 1.0 9.8 ± 0.8

12.5 10/10 19.2 ± 0.4 29.9 ± 0.9 10.6 ± 0.7 103

25 10/10 18.4 ± 0.4 28.7 ± 0.9 10.2 ± 0.7 98

50 10/10 19.2 ± 0.6 29.2 ± 1.3 10.0 ± 0.8 100

100 9/10d 18.7 ± 0.4 28.2 ± 0.9 9.3 ± 0.7 97

a Number of animals surviving at 13 weeks/number initially in group b Weights and weight changes are given as mean ± standard error. Subsequent calculations are based on animals surviving to the end of the

study. Differences from the vehicle control groups were not significant by Dunnettís test. c Week of death: 9

d Week of death: 6
Benzyltrimethylammonium Chloride, NTP TOX 57 31

FIGURE 2 

Body Weights of Mice Administered Benzyltrimethylammonium Chloride 

by Gavage for 13 Weeks
32 Benzyltrimethylammonium Chloride, NTP TOX 57

In male mice, kidney weights were increased in the 50 mg/kg group, and the relative kidney weight was also

increased in the 100 mg/kg group (Table D4). Relative heart weights were increased in the 25 mg/kg or

greater males. However, no chemical-related gross or microscopic lesions were observed (Tables A3 and A4).

Males administered 25 mg/kg or greater had minimally decreased total protein concentrations (Table C4). The

biologic significance of the protein concentration difference was unknown; because the change was minimal

and no other clinical chemistry and pathologic alterations occurred, this difference was not considered to be

clinically significant. No treatment-related differences were detected in reproductive tissue evaluations or

estrous cycle characterizations (Tables E3 and E4). 

GENETIC TOXICOLOGY

Benzyltrimethylammonium chloride (100 to 10,000 μg/plate) was not mutagenic in Salmonella typhimurium

strain TA97, TA98, TA100, or TA1535 with or without induced rat or hamster liver S9 metabolic activation

enzymes (Zeiger et al., 1988; Table F1). Slight toxicity was noted at the two highest concentrations tested in

all four strains. In vivo, benzyltrimethylammonium chloride induced a significant dose-related increase in the

frequency of micronucleated normochromatic erythrocytes in the peripheral blood of male and female mice

administered 12.5 to 100 mg/kg by gavage for 13 weeks (Table F2). Micronucleus analyses yielded positive

trends (P#0.025) for both the male and female data, but only the highest dose tested in males and females

produced an increase in micronuclei that was significantly different from the control frequency (P#0.006).
33

DISCUSSION

Benzyltrimethylammonium chloride is widely used in the chemical, textile, and rubber industries (USEPA,

1990). It was nominated for toxicity testing by the National Institute of Environmental Health Sciences because

of its high production volume, potential for occupational exposure, and the paucity of toxicity information

concerning the chemical.

Based on the doses at which mortality occurred in the 16-day studies, rats and mice appear to be equally

sensitive to benzyltrimethylammonium chloride. On day 1 of the studies, 100% mortality occurred in 125 and

250 mg/kg male and female rats and in 250, 500, and 1,000 mg/kg male and female mice; one of five

125 mg/kg female mice died. The high rate of mortality in rats and mice in the 16-day studies combined with

the absence of an identifiable target organ for benzyltrimethylammonium chloride toxicity suggests that the

cause of death was the result of a pharmacologic effect. The differences in lung and liver weights in

125 mg/kg female mice in the 16-day study were not associated with gross or histologic changes and,

accordingly, were not considered to be related to chemical administration. Benzyltrimethylammonium chloride

mimics the action of acetylcholine by activating the muscarinic and nicotinic receptors and was shown to be

a vasodepressor that could lead to total cardiovascular collapse (Hume and Holland, 1965; Hamilton and

Rubinstein, 1968; Gosselin et al., 1984). The cholinergic activity of benzyltrimethylammonium chloride in

rats in the 16-day study was evidenced by salivation. Benzyltrimethylammonium chloride was four times more

active than acetylcholine in its ability to induce salivation in dogs (Long et al., 1965). However, pupillary

dilation was observed in 63 mg/kg male rats but not in females and therefore was not considered to be related

to chemical administration. Neither salivation nor pupillary constriction occurred in dosed mice. 

The NTP also conducted 14-day dermal studies (unpublished) of benzyltrimethylammonium chloride. Male

and female F344/N rats were administered 0, 11.9, 39.6, or 118.8 mg per day (equivalent to 0, 170, 340, or

680 mg/kg per day for males and 0, 260, 520, or 860 mg/kg per day for females), and B6C3F1 mice were

administered 0, 3.96, 11.9, or 39.6 mg per day (equivalent to 0, 385, 790, or 1,580 mg/kg per day for males

and 0, 450, 900, or 1,800 mg/kg per day for females). Results of these dermal studies were similar to those

of the 16-day gavage studies except that the animals were more sensitive to toxic effects following gavage

administration. Deaths, ataxia, and tremors occurred in rats administered 118.8 mg and mice administered

39.6 mg; these doses were 1.5 to 3.5 orders of magnitude greater than the doses used in the gavage studies.
34 Benzyltrimethylammonium Chloride, NTP TOX 57

This difference in sensitivity is supported by the findings of Sanders et al. (1995), which showed that

benzyltrimethylammonium chloride was poorly absorbed from the skin of rats and mice. 

Based on the mortality in the 16-day studies, doses of 0, 12.5, 25, 50, and 100 mg/kg were administered in

deionized water by gavage to rats and mice in the 13-week studies. Three female rats and one male and one

female mouse died before the end of the studies. There were no significant differences in final mean body

weights of dosed male or female rats or mice compared to the vehicle controls. Because the changes in kidney

and heart weights observed in 50 and 100 mg/kg male mice were not associated with gross or histopathologic

changes, these effects were not considered to be related to chemical administration. Clinical findings and

functional observations in 100 mg/kg rats included eye, nasal, and oral discharges, lacrimation or

chromodacryorrhea, salivation, tremors, pupillary constriction, and impaired coordination. Between 10 and

13 weeks of dosing, female mice displayed increased activity levels immediately following dosing at

100 mg/kg; the activity level returned to normal levels within 1 hour. The cholinergic nature of these effects

suggest an acetylcholine-mimicking activity of benzyltrimethylammonium chloride (Long et al., 1965; Strycker

and Long, 1969).

Significant decreases in total serum protein concentrations were observed in 25 and 50 mg/kg female rats on

day 3, in 25, 50, and 100 mg/kg female rats at week 13, and in 25, 50, and 100 mg/kg male mice at week 13.

The biological significance of this decrease is unknown because the effect was minimal in magnitude and was

not accompanied by other clinical or pathologic alterations. In addition, total serum protein concentrations of

all dosed groups fell within control values reported for rats (7.52 ± 0.20 g/dL) and mice (2.73 ± 0.30 g/dL);

it is therefore unlikely that the decrease was due to chemical administration (Kaneko, 1989). Similarly, the

increase in the mean cell volumes was considered biologically insignificant because the effect was minimal in

magnitude and was not accompanied by hematologic or pathologic alterations. In the 16-day study, edema was

observed in the lung of 250 mg/kg female rats and was considered secondary to lethality induced through

cholinergic stimulation. Lung edema is likely to be the result of a decreased heart rate leading to reduced blood

pressure and the force of contraction prior to death. No histopathologic changes that could be attributed to

benzyltrimethylammonium chloride administration were observed in rats or mice.

Benzyltrimethylammonium chloride was not mutagenic in Salmonella typhimurium strains TA97, TA98,

TA100, and TA1535, with or without S9 metabolic activation enzymes (Zeiger et al., 1988). However, it did

induce significant increases in the frequency of micronucleated normochromatic erythrocytes in peripheral

blood of male and female mice in the 13-week study. Elevated micronucleus frequencies were observed in

male and female mice administered 50 mg/kg or greater, although statistically significant increases were seen
Benzyltrimethylammonium Chloride, NTP TOX 57 35

only at 100 mg/kg. The observation of micronucleus induction suggests that benzyltrimethylammonium

chloride induced chromosomal damage in maturing erythrocytes in the form of breakage and/or mitotic

disruption leading to numerical aberrations (chromosome loss). No alteration in the percentage of

normochromatic erythrocytes in the blood was observed in male or female mice, indicating no overt toxicity

to the bone marrow and no stimulation of erythropoiesis.

Based on the mortality observed in the 16-day and 13-week studies, rats and mice appeared to be equally

sensitive to benzyltrimethylammonium chloride. The minimally toxic dose for rats and mice was estimated to

be 50 mg/kg.
36 Benzyltrimethylammonium Chloride, NTP TOX 57
37

REFERENCES

Aldrich Catalog/Handbook of Fine Chemicals 1990-1991 (1990), p. 146. Aldrich Chemical Company, Inc.,

Milwaukee, WI.

Boorman, G.A., Montgomery, C.A., Jr., Eustis, S.L., Wolfe, M.J., McConnell, E.E., and Hardisty, J.F.

(1985). Quality assurance in pathology for rodent carcinogenicity studies. In Handbook of Carcinogen Testing

(H.A. Milman and E.K. Weisburger, Eds.), pp. 345-357. Noyes Publications, Park Ridge, NJ.

Boorman, G.A., Hickman, R.L., Davis, G.W., Rhodes, L.S., White, N.W., Griffen, T.A., Mayo, J., and

Hamm, T.E., Jr. (1986). Serological titers to murine viruses in 90-day and 2-year studies. In Complications

of Viral and Mycoplasmal Infections in Rodents to Toxicology Research and Testing (T.E. Hamm, Jr., Ed.),

pp. 11-23. Hemisphere Publishing Corporation, Washington, DC.

Chemical Marketing Reporter (April 11, 1983), p. 13.

Code of Federal Regulations (CFR) 21, Part 58.

Dewitt, J.B., Bellack, E., Klingensmith, C.W., Ward, J.C., and Treichler, R. (1953). Relationship Between

Chemical Structure and Toxic Action on Rats. Chemical and Biological Research Center, Review No. 5,

p. 39. National Research Council, Washington, DC.

Dixon, W.J., and Massey, F.J., Jr. (1951). Introduction to Statistical Analysis, 1st ed., pp. 145-147.

McGraw-Hill Book Company, Inc., New York.

Dretchen, K., Diecke, F.P.J., and Long, J.P. (1971). Studies on the nonspecific blocking action of

benzyltrimethylammonium bromide (BTM). J. Pharmacol. Exp. Ther. 177, 369-376.

Dunn, O.J. (1964). Multiple comparisons using rank sums. Technometrics 6, 241-252.
38 Benzyltrimethylammonium Chloride, NTP TOX 57

Dunnett, C.W. (1955). A multiple comparison procedure for comparing several treatments with a control.

J. Am. Stat. Assoc. 50, 1096-1121.

Ellis, K.O., White, R.L., Jr., Wright, G.C., and Wessels, F.L. (1980). Synthesis and skeletal muscle relaxant

activity of quarternary ammonium salts of dantrolene and clodanolene. J. Pharm. Sci. 69, 327-331.

Gart, J.J., Chu, K.C., and Tarone, R.E. (1979). Statistical issues in interpretation of chronic bioassay tests

for carcinogenicity. JNCI 62, 957-974.

Gosselin, R.E., Smith, R.P., Hodge, H.C., and Braddock, J.E. (1984). Clinical Toxicity of Commercial

Products: Acute Poisoning, 5th ed., pp. II-278, III-63 to III-66. Williams and Wilkins, Baltimore, MD.

Hamilton, J.T., and Rubinstein, H.M. (1968). Nicotinic and muscarinic activity of benzyltrimethyl-ammonium

and its alpha-, beta-, and gamma-substituted pyridylmethylammonium analogs. J. Pharmacol. Exp. Ther. 160,

112-123.

Hume, A.S., and Holland, W.C. (1965). Vasopressor and depressor activity of

phenylalkyltrimethylammonium compounds. Arch. Int. Pharmacodyn. 154, 155-160. 

Integrated Laboratory Systems (ILS) (1990). Micronucleus Data Management and Statistical Analysis

Software, Version 1.4. ILS, P.O. Box 13501, Research Triangle Park, NC 72207.

Jonckheere, A.R. (1954). A distribution-free k-sample test against ordered alternatives. Biometrika 41,

133-145.

Kaneko, J.J. (1989). Clinical Biochemistry of Domestic Animals, 4th ed., p. 892. Academic Press, Inc.,

New York.

Karsai, J., SebestyÈn, E., Ga·l, S., G·rdi, I., Siki, K., and KÌss, G. (1986). Plant growth regulating

compositions and process for regulating plant growth. International Patent Application, Patent

No. WO 86/07237.
Benzyltrimethylammonium Chloride, NTP TOX 57 39

Long, J.P., Wong, K.C., and Witt, D.L. (1965). Cholinergic and anticholinergic activity of

benzyltrimethylamine and fluorobenzyl isomers. Arch. Int. Parmacodyn. 155, 282-288.

MacGregor, J.T., Wehr, C.M., Henika, P.R., and Shelby, M.D. (1990). The in vivo erythrocyte

micronucleus test: Measurement at steady state increases assay efficiency and permits integration with toxicity

studies. Fundam. Appl. Toxicol. 14, 513-522.

Maronpot, R.R., and Boorman, G.A. (1982). Interpretation of rodent hepatocellular proliferative alterations

and hepatocellular tumors in chemical safety assessment. Toxicol. Pathol. 10, 71-80.

Moore, S.B., Diehl, R.A., Barnhardt, J.M., and Avery, G.B. (1987). Aquatic toxicities of textile surfactants.

Text. Chemist Colorist 19, 29-32.

Morrison, D.F. (1976). Multivariate Statistical Methods, 2nd ed., pp. 170-179. McGraw-Hill Book

Company, New York.

National Institute for Occupational Safety and Health (NIOSH) (1990). National Occupational Exposure

Survey (1981-1983), unpublished provisional data as of July 1, 1990. NIOSH, Cincinnati, OH.

National Toxicology Program (NTP) (1991). Technical Protocol for Sperm Morphology and Vaginal Cytology

Evaluations in Toxicity Testing for Rats and Mice, 10/31/82 version (updated May 1991). Research Triangle

Park, NC.

Neef, C., Oosting, R., and Meijer, D.K.F. (1984). Structure-pharmacokinetics relationship of quaternary

ammonium compounds. Elimination and distribution characteristics. Naunyn Schmiedebergís Arch.

Pharmacol. 328, 103-110.

Rao, G.N., Haseman, J.K., and Edmondson, J. (1989a). Influence of viral infections on body weight,

survival, and tumor prevalence in Fischer 344/NCr rats on two-year studies. Lab. Anim. Sci. 39, 389-393.

Rao, G.N., Piegorsch, W.W., Crawford, D.D., Edmondson, J., and Haseman, J.K. (1989b). Influence of

viral infections on body weight, survival, and tumor prevalence of B6C3F1 (C57BL/6N × C3H/HeN) mice

in carcinogenicity studies. Fundam. Appl. Toxicol. 13, 156-164.
40 Benzyltrimethylammonium Chloride, NTP TOX 57

Sanders, M., Griffin, R.J., Burka, L.T., and Matthews, H.B. (1995). Toxicokinetics of the cholinomimetic

compound benzyltrimethylammonium chloride in the male rat and mouse. Xenobiotica 25, 303-313.

Sax, N.I., and Lewis, R.J., Sr. (1987). Hawleyís Condensed Chemical Dictionary, 11th ed., p. 138.

Van Nostrand Reinhold, New York.

Sax, N.I., and Lewis, R.J., Sr. (1989). Dangerous Properties of Industrial Materials, 7th ed., p. 426.

Van Nostrand Reinhold, New York.

Shirley, E. (1977). A non-parametric equivalent of Williamsí test for contrasting increasing dose levels of a

treatment. Biometrics 33, 386-389.

The Sigma-Aldrich Library of Chemical Safety Data (1988). 2nd ed. (R.E. Lenga, Ed.). Sigma-Aldrich

Corp., Milwaukee, WI. 

Strycker, S.J., and Long, J.P. (1969). Studies on the muscarinic and antimuscarinic activity of

benzyltrimethylammonium bromide (BTM). J. Pharm. Sci. 58, 671-675.

United States Environmental Protection Agency (USEPA) (1990). U.S. Environmental Protection Agency,

Computer Printout (TSCAPP): 1983 Production Statistics for Chemicals in the Non-confidential TSCA

Chemical Substances Inventory. Office of Pesticides and Toxic Substances, Washington, DC.

United States International Trade Commission (USITC) (1986). Synthetic Organic Chemicals, United States

Production and Sales, 1986, pp. 145, 167, 177-178. U.S. Publication No. 2009. Government Printing Office,

Washington, DC. 

United States International Trade Commission (USITC) (1987). Synthetic Organic Chemicals, United States

Production and Sales, 1987, pp. 3-4, 12-6. Publication No. 2118. U.S. Government Printing Office,

Washington, DC. 

United States International Trade Commission (USITC) (1989). Synthetic Organic Chemicals, United States

Production and Sales, 1988, pp. 12-6, 12-27, A-5, A-14 to A-18. Publication No. 2219. U.S. Government

Printing Office, Washington, DC.
Benzyltrimethylammonium Chloride, NTP TOX 57 41

Weast, R.C., Ed. (1989). CRC Handbook of Chemistry and Physics (1988-1989), 69th ed., p. C-150. CRC

Press, Inc., Boca Raton, FL.

Williams, D.A. (1971). A test for differences between treatment means when several dose levels are compared

with a zero dose control. Biometrics 27, 103-117.

Williams, D.A. (1972). The comparison of several dose levels with a zero dose control. Biometrics 28,

519-531.

Zeiger, E., Anderson, B., Haworth, S., Lawlor, T., and Mortelmans, K. (1988). Salmonella mutagenicity

tests: IV. Results from the testing of 300 chemicals. Environ. Mol. Mutagen. 11 (Suppl. 12), 1-158.
42 Benzyltrimethylammonium Chloride, NTP TOX 57
A-1

APPENDIX A

SUMMARY OF NONNEOPLASTIC LESIONS

IN RATS AND MICE

TABLE A1 Summary of the Incidence of Nonneoplastic Lesions in Male Rats 

in the 13-Week Gavage Study of Benzyltrimethylammonium Chloride . . . . . . . . . . . . A-2

TABLE A2 Summary of the Incidence of Nonneoplastic Lesions in Female Rats 

in the 13-Week Gavage Study of Benzyltrimethylammonium Chloride . . . . . . . . . . . . A-4

TABLE A3 Summary of the Incidence of Nonneoplastic Lesions in Male Mice 

in the 13-Week Gavage Study of Benzyltrimethylammonium Chloride . . . . . . . . . . . . A-6

TABLE A4 Summary of the Incidence of Nonneoplastic Lesions in Female Mice 

in the 13-Week Gavage Study of Benzyltrimethylammonium Chloride . . . . . . . . . . . . A-8
A-2 Benzyltrimethylammonium Chloride, NTP TOX 57

TABLE A1

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 13-Week Gavage Study 

of Benzyltrimethylammonium Chloridea

Vehicle Control 12.5 mg/kg 25 mg/kg 50 mg/kg 100 mg/kg

Disposition Summary

Animals initially in study 10 10 10 10 10

Survivors

Terminal sacrifice 10 10 10 10 10

Animals examined microscopically 10 2 1 10 10

Alimentary System

Liver (10) (2) (10)

Inflammation, focal 4 (40%) 5 (50%)

Hepatocyte, centrilobular, 

 vacuolization cytoplasmic 1 (10%)

Cardiovascular System

Heart (10) (10)

Inflammation, focal 8 (80%) 5 (50%)

Endocrine System

Pituitary gland (10) (10)

Cyst 1 (10%) 1 (10%) 

Thyroid gland (10) (10)

Follicle, cyst 2 (20%) 1 (10%) 

General Body System

None

Genital System

Epididymis (10) (1) (10)

Inflammation, focal, 

 granulomatous 1 (100%) 

Prostate (10) (10)

Infiltration cellular, focal, 

 lymphocyte 1 (10%)

Inflammation 1 (10%)

Testes (10) (1) (10)

Atrophy 1 (100%)

Hematopoietic System

Spleen (10) (10)

Hematopoietic cell proliferation 3 (30%) 4 (40%) 

Integumentary System

None
Benzyltrimethylammonium Chloride, NTP TOX 57 A-3

TABLE A1

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 13-Week Gavage Study 

of Benzyltrimethylammonium Chloride

 Vehicle Control 12.5 mg/kg 25 mg/kg 50 mg/kg 100 mg/kg

Musculoskeletal System

None

Nervous System

None

Respiratory System

Lung (10) (10) (10)

Inflammation, focal 6 (60%) 1 (10%) 5 (50%) 

Nose (10) (10)

Inflammation 2 (20%)

Special Senses System

None

Urinary System

Kidney (10) (1) (10)

Renal tubule, hemorrhage, focal 1 (10%)

a Number of animals examined microscopically at the site and the number of animals with lesion
A-4 Benzyltrimethylammonium Chloride, NTP TOX 57

TABLE A2

Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 13-Week Gavage Study 

of Benzyltrimethylammonium Chloridea

Vehicle Control 12.5 mg/kg 25 mg/kg 50 mg/kg 100 mg/kg

Disposition Summary

Animals initially in study 10 10 10 10 10

Early death

Accidental death 1

Natural deaths 2

Survivors

Died last week of study 1

Terminal sacrifice 10 10 9 9 8

Animals examined microscopically 10 3 2 10 10

Alimentary System

Liver (10) (1) (1) (10)

Inflammation, focal 5 (50%) 4 (40%)

Hepatocyte, centrilobular,

 vacuolization cytoplasmic 1 (100%)

Mesentery (1) (1)

Fat, necrosis 1 (100%) 1 (100%) 

Pancreas (10) (1) (10)

Acinus, atrophy 1 (10%) 

Acinus, degeneration, focal 1 (10%)

Cardiovascular System

Heart (10) (1) (10)

Inflammation, focal 5 (50%) 1 (100%) 4 (40%) 

Endocrine System

Pituitary gland (10) (10)

Pars distalis, cyst 1 (10%)

Thyroid gland (10) (1) (10)

Follicle, cyst 1 (10%)

General Body System

None

Genital System

Uterus (10) (2) (1) (1) (10)

Bilateral, cyst 2 (100%) 1 (100%)

Bilateral, dilatation 2 (20%) 2 (20%) 

Hematopoietic System

Spleen (10) (1) (10)

Hematopoietic cell proliferation 1 (10%) 1 (100%)
Benzyltrimethylammonium Chloride, NTP TOX 57 A-5

TABLE A2

Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 13-Week Gavage Study 

of Benzyltrimethylammonium Chloride

Vehicle Control 12.5 mg/kg 25 mg/kg 50 mg/kg 100 mg/kg

Integumentary System

None

Musculoskeletal System

None

Nervous System

None

Respiratory System

Lung (10) (1) (10) (10)

Congestion 1 (10%) 

Inflammation, focal 3 (30%) 1 (100%) 3 (30%) 6 (60%) 

Nose (10) (10)

Inflammation, chronic active 1 (10%) 

Special Senses System

None

Urinary System

None

a Number of animals examined microscopically at the site and the number of animals with lesion
A-6 Benzyltrimethylammonium Chloride, NTP TOX 57

TABLE A3

Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 13-Week Gavage Study 

of Benzyltrimethylammonium Chloridea

Vehicle Control 12.5 mg/kg 25 mg/kg 50 mg/kg 100 mg/kg

Disposition Summary

Animals initially in study 10 10 10 10 10

Early death

Natural death 1

Survivors

Died last week of study 1

Terminal sacrifice 10 10 10 9 9

Animals examined microscopically 10 10

Alimentary System

Liver (10) (10)

Inflammation, chronic, focal 1 (10%) 

Hepatocyte, centrilobular, 

 hypertrophy 3 (30%) 3 (30%) 

Salivary glands (10) (10)

Infiltration cellular, focal, 

 lymphocyte 1 (10%) 

Cardiovascular System

None

Endocrine System

Adrenal cortex (10) (10)

Capsule, hyperplasia, focal 1 (10%) 

General Body System

None

Genital System

Prostate (10) (10)

Infiltration cellular, lymphocyte 1 (10%)

Hematopoietic System

Spleen (10) (10)

Hematopoietic cell proliferation 1 (10%) 1 (10%) 

Lymphoid follicle, hyperplasia 2 (20%)

Integumentary System

None

Musculoskeletal System

None
Benzyltrimethylammonium Chloride, NTP TOX 57 A-7

TABLE A3

Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 13-Week Gavage Study

of Benzyltrimethylammonium Chloride

Vehicle Control 12.5 mg/kg 25 mg/kg 50 mg/kg 100 mg/kg

Nervous System

None

Respiratory System

None

Special Senses System

None

Urinary System

None

a Number of animals examined microscopically at the site and the number of animals with lesion
A-8 Benzyltrimethylammonium Chloride, NTP TOX 57

TABLE A4

Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 13-Week Gavage Study

of Benzyltrimethylammonium Chloridea

Vehicle Control 12.5 mg/kg 25 mg/kg 50 mg/kg 100 mg/kg

Disposition Summary

Animals initially in study 10 10 10 10 10

Early death

Natural death 1

Survivors

Terminal sacrifice 10 10 10 10 9

Animals examined microscopically 10 10

Alimentary System

Liver (10) (10)

Inflammation, chronic, focal 1 (10%)

Necrosis, focal 2 (20%) 1 (10%) 

Hepatocyte, centrilobular, 

 hypertrophy 3 (30%) 3 (30%) 

Cardiovascular System

None

Endocrine System

Adrenal cortex (10) (10)

Bilateral, capsule, hyperplasia, 

 focal 5 (50%) 5 (50%)

Capsule, hyperplasia, focal 1 (10%)

Thyroid gland (10) (10)

Ultimobranchial cyst 1 (10%) 

General Body System

None

Genital System

Ovary (10) (10)

Hemorrhage 1 (10%) 

Uterus (10) (10)

Endometrium, hyperplasia, cystic 3 (30%)

Hematopoietic System

Lymph node, mandibular (10) (10)

Hemorrhage 1 (10%) 

Hyperplasia, lymphoid 1 (10%)

Lymph node, mesenteric (10) (10)

Hyperplasia, lymphoid 1 (10%)

Spleen (10) (10)

Hematopoietic cell proliferation 1 (10%) 3 (30%)
Benzyltrimethylammonium Chloride, NTP TOX 57 A-9

TABLE A4

Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 13-Week Gavage Study

of Benzyltrimethylammonium Chloride

Vehicle Control 12.5 mg/kg 25 mg/kg 50 mg/kg 100 mg/kg

Integumentary System

None

Musculoskeletal System

None

Nervous System

None

Respiratory System

Lung (10) (10)

Interstitium, inflammation,

 chronic 1 (10%) 2 (20%) 

Nose (10) (10)

Olfactory epithelium, cytoplasmic

 alteration 1 (10%) 

Special Senses System

None

Urinary System

Urinary bladder (10) (10)

Infiltration cellular, lymphocyte 1 (10%)

a Number of animals examined microscopically at the site and the number of animals with lesion
A-10 Benzyltrimethylammonium Chloride, NTP TOX 57
B-1

APPENDIX B

FUNCTIONAL OBSERVATION BATTERY RESULTS

TABLE B1 Selected Functional Observation Battery Data for Rats in the 16-Day Gavage Study

of Benzyltrimethylammonium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-2

TABLE B2 Functional Observation Battery Data for Rats in the 13-Week Gavage Study

of Benzyltrimethylammonium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-3
B-2 Benzyltrimethylammonium Chloride, NTP TOX 57

TABLE B1

Selected Functional Observation Battery Data for Rats in the 16-Day Gavage Study

of Benzyltrimethylammonium Chloridea

Vehicle

Control 16 mg/kg 32 mg/kg 63 mg/kg

Male

Activity

Minimum exploratory movements 1/5 1/5 0/5 1/5

Somewhat high 0/5 0/5 1/5 0/5

Pupillary constriction or dilation

Dilated 0/5 0/5 1/5 3/5

Salivation

Slight 0/5 0/5 0/5 4/5*

Severe 0/5 0/5 0/5 1/5

Tremors

Mild 0/5 0/5 0/5 2/5

Female

Salivation

Slight 0/5 1/5 0/5 2/5

* Significantly different (P#0.05) from the vehicle control group by the Fisher exact test a Number of animals with behavior/number of animals observed
Benzyltrimethylammonium Chloride, NTP TOX 57 B-3

TABLE B2

Functional Observation Battery Data for Rats in the 13-Week Gavage Study

of Benzyltrimethylammonium Chloridea

Vehicle

Control 12.5 mg/kg 25 mg/kg 50 mg/kg 100 mg/kg

Male

Activity

Day 10

Minimum exploratory

 movements 2/10 1/10 1/10 3/10 1/10

Normal 8/10 9/10 9/10 7/10 9/10

Day 85

Low 0/10 0/10 0/10 0/10 2/10

Minimum exploratory

 movements 4/10 0/10* 1/10 0/10* 1/10

Normal 6/10 10/10* 9/10 10/10* 7/10

Backward walking

Day 10

Not present 10/10 10/10 10/10 10/10 10/10

Day 85

Not present 10/10 10/10 10/10 10/10 10/10

Body position

Day 10

Sitting/standing (normal) 10/10 10/10 10/10 10/10 10/10

Rearing (normal) 8/10 10/10 9/10 8/10 8/10

Day 85

Sitting/standing (normal) 10/10 10/10 10/10 10/10 8/10

Rearing (normal) 10/10 10/10 8/10 10/10 5/10*

Crouched over 0/10 0/10 0/10 0/10 2/10

Head bobbing 0/10 0/10 0/10 0/10 2/10

Circling

Day 10

Not present 10/10 10/10 10/10 10/10 10/10

Day 85

Not present 10/10 10/10 10/10 10/10 10/10

Compulsive biting or licking

Day 10

Normal 10/10 10/10 10/10 10/10 10/10

Day 85

Normal 10/10 10/10 10/10 10/10 10/10

Convulsions

Day 10

None 10/10 10/10 10/10 10/10 10/10

Day 85

None 10/10 10/10 10/10 10/10 8/10

Mild 0/10 0/10 0/10 0/10 1/10

Severe 0/10 0/10 0/10 0/10 1/10

Coordination of movement

Day 10

Normal 10/10 10/10 10/10 10/10 10/10

Day 85

Normal 10/10 10/10 10/10 10/10 8/10

Moderately impaired 0/10 0/10 0/10 0/10 1/10

Severely impaired 0/10 0/10 0/10 0/10 1/10
B-4 Benzyltrimethylammonium Chloride, NTP TOX 57

TABLE B2

Functional Observation Battery Data for Rats in the 13-Week Gavage Study

of Benzyltrimethylammonium Chloride

Vehicle

Control 12.5 mg/kg 25 mg/kg 50 mg/kg 100 mg/kg

Male (continued)

Diarrhea

Day 10

Not present 10/10 10/10 10/10 10/10 10/10

Day 85

Not present 10/10 10/10 10/10 10/10 10/10

Gait

Day 10

Normal 10/10 10/10 10/10 10/10 10/10

Day 85

Normal 10/10 10/10 10/10 10/10 7/10

Ataxia 0/10 0/10 0/10 0/10 1/10

Hind limbs impaired 0/10 0/10 0/10 0/10 1/10

Hunched or crouched 0/10 0/10 0/10 0/10 2/10

Body drags/is flattened 0/10 0/10 0/10 0/10 2/10

General behavior

Day 10

Normal 10/10 10/10 10/10 10/10 10/10

Day 85

Normal 10/10 10/10 10/10 10/10 8/10

Abnormal 0/10 0/10 0/10 0/10 2/10

Head flick

Day 10

Normal 10/10 10/10 10/10 10/10 10/10

Day 85

Normal 10/10 10/10 10/10 10/10 9/10

Mild 0/10 0/10 0/10 0/10 1/10

Head search

Day 10

Normal 10/10 10/10 10/10 10/10 10/10

Day 85

Normal 10/10 10/10 10/10 10/10 10/10

Lacrimation or chromodacryorrhea

Day 10

None 10/10 10/10 10/10 10/10 9/10

Slight 0/10 0/10 0/10 0/10 1/10

Day 85

None 10/10 10/10 10/10 10/10 4/10**

Slight 0/10 0/10 0/10 0/10 5/10*

Severe 0/10 0/10 0/10 0/10 1/10

Piloerection

Day 10

Not present 10/10 10/10 10/10 10/10 10/10

Day 85

Present 0/10 0/10 0/10 0/10 1/10

Not present 10/10 10/10 10/10 10/10 9/10

Pupillary constriction or dilation

Day 10

Normal 10/10 10/10 10/10 10/10 10/10

Day 85

Normal 10/10 10/10 10/10 10/10 9/10

Constricted 0/10 0/10 0/10 0/10 1/10
Benzyltrimethylammonium Chloride, NTP TOX 57 B-5

TABLE B2

Functional Observation Battery Data for Rats in the 13-Week Gavage Study

of Benzyltrimethylammonium Chloride

Vehicle

Control 12.5 mg/kg 25 mg/kg 50 mg/kg 100 mg/kg

Male (continued)

Respiration

Day 10

Normal 10/10 10/10 10/10 10/10 10/10

Day 85

Normal 10/10 10/10 10/10 10/10 9/10

Labored 0/10 0/10 0/10 0/10 1/10

Salivation

Day 10

None 9/10 10/10 10/10 9/10 7/10

Slight 1/10 0/10 0/10 1/10 2/10

Severe 0/10 0/10 0/10 0/10 1/10

Day 85

None 10/10 9/10 8/10 4/10** 0/10**

Slight 0/10 1/10 2/10 4/10* 2/10

Severe 0/10 0/10 0/10 2/10 8/10**

Self mutilation

Day 10

Not present 10/10 10/10 10/10 10/10 10/10

Day 85

Not present 10/10 10/10 10/10 10/10 10/10

Tremors

Day 10

Normal 10/10 10/10 10/10 10/10 10/10

Day 85

Normal 10/10 10/10 10/10 10/10 5/10*

Mild 0/10 0/10 0/10 0/10 2/10

Severe whole body 0/10 0/10 0/10 0/10 3/10

Urination

Day 10

Normal 10/10 10/10 10/10 10/10 10/10

Day 85

Normal 10/10 10/10 10/10 10/10 10/10

Vocalization

Day 10

Not present 10/10 10/10 10/10 10/10 10/10

Day 85

Not present 10/10 10/10 10/10 10/10 10/10

Female

Activity

Day 10

Minimum exploratory

 movements 0/10 4/10* 2/10 3/10 1/10

Normal 10/10 6/10* 8/10 7/10 9/10

Day 85

Minimum exploratory

 movements 1/10 5/10 2/9 1/10 3/8

Normal 9/10 5/10 7/9 9/10 5/8
B-6 Benzyltrimethylammonium Chloride, NTP TOX 57

TABLE B2

Functional Observation Battery Data for Rats in the 13-Week Gavage Study

of Benzyltrimethylammonium Chloride

Vehicle

Control 12.5 mg/kg 25 mg/kg 50 mg/kg 100 mg/kg

Female (continued)

Backward walking

Day 10

Not present 10/10 10/10 10/10 10/10 10/10

Day 85

Not present 10/10 10/10 9/9 10/10 8/8

Body position

Day 10

Sitting/standing (normal) 10/10 10/10 10/10 10/10 10/10

Rearing (normal) 10/10 8/10 8/10 4/10** 8/10

Day 85

Sitting/standing (normal) 10/10 10/10 9/9 10/10 7/8

Rearing (normal) 10/10 8/10 7/9 9/10 5/8

Flattened 0/10 0/10 0/9 0/10 1/8

Crouched over 0/10 0/10 0/9 0/10 2/8

Circling

Day 10

Not present 10/10 10/10 10/10 10/10 10/10

Day 85

Not present 10/10 10/10 9/9 10/10 8/8

Compulsive biting or licking

Day 10

Normal 10/10 10/10 10/10 10/10 10/10

Day 85

Normal 10/10 10/10 9/9 10/10 8/8

Convulsions

Day 10

None 10/10 10/10 10/10 10/10 10/10

Day 85

None 10/10 10/10 9/9 10/10 8/8

Coordination of movement

Day 10

Normal 10/10 10/10 10/10 10/10 10/10

Day 85

Normal 10/10 10/10 9/9 10/10 5/8

Slightly impaired 0/10 0/10 0/9 0/10 3/8

Diarrhea

Day 10

Not present 10/10 10/10 10/10 10/10 10/10

Day 85

Not present 10/10 10/10 9/9 10/10 8/8

Gait

Day 10

Normal 10/10 10/10 10/10 10/10 10/10

Day 85

Normal 10/10 10/10 9/9 10/10 4/8*

Ataxia 0/10 0/10 0/9 0/10 2/8

Hind limbs impaired 0/10 0/10 0/9 0/10 2/8

Forelimbs drag 0/10 0/10 0/9 0/10 1/8

Walks on tiptoes 0/10 0/10 0/9 0/10 1/8

Hunched or crouched 0/10 0/10 0/9 0/10 1/8

Body drags/is flattened 0/10 0/10 0/9 0/10 1/8
Benzyltrimethylammonium Chloride, NTP TOX 57 B-7

TABLE B2

Functional Observation Battery Data for Rats in the 13-Week Gavage Study

of Benzyltrimethylammonium Chloride

Vehicle

Control 12.5 mg/kg 25 mg/kg 50 mg/kg 100 mg/kg

Female (continued)

General behavior

Day 10

Normal 10/10 10/10 10/10 10/10 10/10

Day 85

Normal 10/10 10/10 9/9 9/10 8/8

Abnormal 0/10 0/10 0/9 1/10 0/8

Head flick

Day 10

Normal 10/10 10/10 10/10 10/10 10/10

Day 85

Normal 10/10 10/10 9/9 10/10 7/8

Mild 0/10 0/10 0/9 0/10 1/8

Head search

Day 10

Normal 10/10 10/10 10/10 10/10 10/10

Day 85

Normal 10/10 10/10 9/9 10/10 8/8

Lacrimation or chromodacryorrhea

Day 10

None 9/10 10/10 9/10 10/10 10/10

Slight 1/10 0/10 1/10 0/10 0/10

Day 85

None 10/10 7/10 6/9 6/10* 2/8**

Slight 0/10 3/10 3/9 4/10* 6/8**

Piloerection

Day 10

Not present 10/10 10/10 10/10 10/10 10/10

Day 85

Not present 10/10 10/10 9/9 10/10 8/8

Pupillary constriction or dilation

Day 10

Normal 10/10 10/10 9/10 10/10 10/10

Dilated 0/10 0/10 1/10 0/10 0/10

Day 85

Normal 10/10 10/10 9/9 7/10 4/8*

Constricted 0/10 0/10 0/9 3/10 4/8*

Respiration

Day 10

Normal 10/10 10/10 10/10 10/10 10/10

Day 85

Normal 10/10 10/10 9/9 10/10 8/8

Salivation

Day 10

None 10/10 10/10 10/10 10/10 9/10

Slight 0/10 0/10 0/10 0/10 1/10

Day 85

None 9/10 9/10 8/9 9/10 4/8

Slight 1/10 1/10 1/9 0/10 3/8

Severe 0/10 0/10 0/9 1/10 1/8
B-8 Benzyltrimethylammonium Chloride, NTP TOX 57

TABLE B2

Functional Observation Battery Data for Rats in the 13-Week Gavage Study

of Benzyltrimethylammonium Chloride

Vehicle

Control 12.5 mg/kg 25 mg/kg 50 mg/kg 100 mg/kg

Female (continued)

Self mutilation

Day 10

Not present 10/10 10/10 10/10 10/10 10/10

Day 85

Not present 10/10 10/10 9/9 10/10 8/8

Tremors

Day 10

Normal 10/10 10/10 10/10 10/10 10/10

Day 85

Normal 10/10 10/10 9/9 10/10 3/8**

Mild 0/10 0/10 0/9 0/10 4/8*

Mild whole body 0/10 0/10 0/9 0/10 1/8

Urination

Day 10

Normal 10/10 10/10 10/10 10/10 10/10

Day 85

Normal 10/10 10/10 9/9 10/10 8/8

Vocalization

Day 10

Not present 10/10 10/10 10/10 10/10 10/10

Day 85

Not present 10/10 10/10 9/9 10/10 8/8

* Significantly different (P#0.05) from the vehicle control group by the Fisher exact test

** P#0.01 a Number of animals with behavior/number of animals observed
C-1

APPENDIX C

HEMATOLOGY AND CLINICAL CHEMISTRY

RESULTS

TABLE C1 Hematology and Clinical Chemistry Data for Rats in the 16-Day Gavage Study

of Benzyltrimethylammonium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-2

TABLE C2 Hematology and Clinical Chemistry Data for Rats in the 13-Week Gavage Study 

of Benzyltrimethylammonium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-4

TABLE C3 Hematology and Clinical Chemistry Data for Mice in the 16-Day Gavage Study

of Benzyltrimethylammonium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-10

TABLE C4 Clinical Chemistry Data for Mice in the 13-Week Gavage Study

of Benzyltrimethylammonium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-12
C-2 Benzyltrimethylammonium Chloride, NTP TOX 57

TABLE C1

Hematology and Clinical Chemistry Data for Rats in the 16-Day Gavage Study

of Benzyltrimethylammonium Chloridea

Vehicle Control 16 mg/kg 32 mg/kg 63 mg/kg

Male

n 5 5 5 5

Hematology

Automated hematocrit (%) 40.4 ± 0.6 40.3 ± 0.6 40.7 ± 1.0 40.3 ± 0.4

Manual hematocrit (%) 45.4 ± 0.7 45.8 ± 0.7 45.8 ± 1.2 45.4 ± 0.5

Hemoglobin (g/dL) 15.0 ± 0.3 15.1 ± 0.1 15.1 ± 0.3 14.8 ± 0.1

Erythrocytes (106

/μL) 7.06 ± 0.10 7.08 ± 0.12 7.13 ± 0.20 6.99 ± 0.11

Reticulocytes (106

/μL) 0.29 ± 0.02 0.24 ± 0.06 0.25 ± 0.04 0.46 ± 0.13

Nucleated erythrocytes/100 leukocytes 0.40 ± 0.25 0.40 ± 0.25 0.20 ± 0.20 0.40 ± 0.25

Mean cell volume (fL) 57.1 ± 0.2 56.9 ± 0.3 57.1 ± 0.3 57.6 ± 0.5

Mean cell hemoglobin (pg) 21.2 ± 0.1 21.3 ± 0.3 21.3 ± 0.2 21.3 ± 0.2

Mean cell hemoglobin

 concentration (g/dL) 37.1 ± 0.2 37.4 ± 0.5 37.3 ± 0.3 36.8 ± 0.2

Platelets (103

/μL) 838.4 ± 15.1 820.0 ± 34.2 857.4 ± 21.1 774.0 ± 9.4

Leukocytes (103

/μL) 9.10 ± 0.32 8.42 ± 0.18 8.88 ± 0.55 9.06 ± 0.40

Segmented neutrophils (103

/μL) 0.86 ± 0.16 0.82 ± 0.14 0.82 ± 0.14 1.00 ± 0.14

Lymphocytes (103

/μL) 8.06 ± 0.31 7.38 ± 0.26 7.91 ± 0.35 7.63 ± 0.26

Monocytes (103

/μL) 0.07 ± 0.05 0.20 ± 0.09 0.10 ± 0.06 0.37 ± 0.12

Eosinophils (103

/μL) 0.12 ± 0.06 0.02 ± 0.02 0.06 ± 0.04 0.06 ± 0.04

Clinical Chemistry

Urea nitrogen (mg/dL) 24.0 ± 0.5 25.8 ± 0.4 23.0 ± 0.7 25.0 ± 0.5

Creatinine (mg/dL) 0.70 ± 0.00 0.70 ± 0.00 0.70 ± 0.00 0.70 ± 0.00

Total protein (g/dL) 6.6 ± 0.2 6.3 ± 0.1 6.6 ± 0.1 6.2 ± 0.1

Albumin (g/dL) 4.8 ± 0.1 4.7 ± 0.1 4.7 ± 0.1 4.6 ± 0.1

Alanine aminotransferase (IU/L) 48 ± 1 56 ± 4 43 ± 1 43 ± 4

Alkaline phosphatase (IU/L) 545 ± 15 553 ± 13 566 ± 16 559 ± 13

Creatine kinase (IU/L) 440 ± 81 542 ± 147 505 ± 81 444 ± 79

Sorbitol dehydrogenase (IU/L) 33 ± 2 32 ± 3 31 ± 2 32 ± 2

Serum cholinesterase (IU/L) 676.8 ± 28.8 753.2 ± 13.5 738.4 ± 23.4 789.0 ± 39.7*

Erythrocyte cholinesterase (IU/L) 530.2 ± 143.6 726.4 ± 113.2 694.4 ± 121.9 726.6 ± 71.4

Bile acids (μmol/L) 28.3 ± 4.5 28.6 ± 4.0 39.9 ± 7.2 34.5 ± 5.1
Benzyltrimethylammonium Chloride, NTP TOX 57 C-3

TABLE C1

Hematology and Clinical Chemistry Data for Rats in the 16-Day Gavage Study

of Benzyltrimethylammonium Chloride

Vehicle Control 16 mg/kg 32 mg/kg 63 mg/kg

Female

n 5555

Hematology 

Automated hematocrit (%) 44.5 ± 0.4 42.6 ± 0.8 42.5 ± 0.4 44.2 ± 1.1 

Manual hematocrit (%) 46.4 ± 0.4 45.0 ± 0.5 44.8 ± 0.4 46.2 ± 0.9

Hemoglobin (g/dL) 15.6 ± 0.1 15.2 ± 0.2 15.0 ± 0.1 15.7 ± 0.3 

Erythrocytes (106

/μL) 7.59 ± 0.08 7.24 ± 0.13 7.20 ± 0.06 7.58 ± 0.20 

Reticulocytes (106

/μL) 0.20 ± 0.03 0.22 ± 0.03 0.18 ± 0.02 0.23 ± 0.03 

Nucleated erythrocytes/100 leukocytes 0.00 ± 0.00 0.80 ± 0.37 0.20 ± 0.20 0.00 ± 0.00 

Mean cell volume (fL) 58.6 ± 0.2 58.9 ± 0.1 59.1 ± 0.2 58.3 ± 0.3 

Mean cell hemoglobin (pg) 20.6 ± 0.2 21.0 ± 0.2 20.8 ± 0.1 20.7 ± 0.2 

Mean cell hemoglobin 

 concentration (g/dL) 35.2 ± 0.3 35.7 ± 0.4 35.2 ± 0.3 35.5 ± 0.2 

Platelets (103

/μL) 714.8 ± 13.5 755.2 ± 23.9 763.2 ± 14.2 710.0 ± 23.9 

Leukocytes (103

/μL) 9.28 ± 0.50 9.46 ± 0.19 9.38 ± 0.93 8.24 ± 0.30 

Segmented neutrophils (103

/μL) 1.30 ± 0.26 1.21 ± 0.22 1.17 ± 0.23 0.77 ± 0.18 

Lymphocytes (103

/μL) 7.72 ± 0.37 7.89 ± 0.27 8.05 ± 0.84 7.32 ± 0.44 

Monocytes (103

/μL) 0.19 ± 0.05 0.27 ± 0.05 0.13 ± 0.04 0.09 ± 0.05 

Eosinophils (103

/μL) 0.07 ± 0.03 0.10 ± 0.03 0.04 ± 0.02 0.06 ± 0.02 

Clinical Chemistry

Urea nitrogen (mg/dL) 23.8 ± 0.4 23.6 ± 0.2 22.6 ± 1.3 22.4 ± 0.8 

Creatinine (mg/dL) 0.70 ± 0.00 0.68 ± 0.02 0.66 ± 0.02 0.66 ± 0.04 

Total protein (g/dL) 6.0 ± 0.1 6.0 ± 0.1 5.8 ± 0.1 6.0 ± 0.1

Albumin (g/dL) 4.5 ± 0.1 4.5 ± 0.1 4.3 ± 0.1 4.4 ± 0.1 

Alanine aminotransferase (IU/L) 40 ± 2 37 ± 1 37 ± 2 37 ± 1 

Alkaline phosphatase (IU/L) 505 ± 8 490 ± 18 501 ± 31 518 ± 9 

Creatine kinase (IU/L) 432 ± 61 272 ± 32 421 ± 70 378 ± 86 

Sorbitol dehydrogenase (IU/L) 30 ± 1 28 ± 0 27 ± 2 26 ± 1

Serum cholinesterase (IU/L) 2,364.0 ± 120.6 2,577.8 ± 189.9 2,061.0 ± 123.8 2,138.4 ± 223.7 

Erythrocyte cholinesterase (IU/L) 673.8 ± 175.9 400.6 ± 164.4 1,185.8 ± 193.3 819.2 ± 204.7 

Bile acids (μmol/L) 29.2 ± 2.9 31.4 ± 5.2 28.5 ± 5.0 28.9 ± 6.2

* Significantly different (P#0.05) from the vehicle control group by Dunnís test a Mean ± standard error. Statistical tests were performed on unrounded data. No data are available for the 125 and 250 mg/kg groups due

to 100% mortality.
C-4 Benzyltrimethylammonium Chloride, NTP TOX 57

TABLE C2

Hematology and Clinical Chemistry Data for Rats in the 13-Week Gavage Study 

of Benzyltrimethylammonium Chloridea

Vehicle Control 12.5 mg/kg 25 mg/kg 50 mg/kg 100 mg/kg

Male

n

Day 3 10 10 10 10 10

Day 21 10 10 10 10 10

Week 13 10 9 10 10 10

Hematology

Automated hematocrit (%)

Day 3 38.4 ± 0.5 39.0 ± 0.6 37.9 ± 0.4 38.7 ± 0.4 39.1 ± 0.7

Day 21 44.3 ± 0.4 44.3 ± 0.4 44.1 ± 0.3 44.8 ± 0.5 43.5 ± 0.7

Week 13 46.8 ± 0.4 48.5 ± 0.3* 47.9 ± 0.5 46.5 ± 0.5 46.5 ± 0.4

Manual hematocrit (%) 

Day 3 42.2 ± 0.7 41.5 ± 0.7 41.2 ± 0.4 43.1 ± 0.4 42.5 ± 0.8

Day 21 47.7 ± 0.5 47.5 ± 0.7 47.0 ± 0.5 48.0 ± 0.6 47.6 ± 0.6

Week 13 48.9 ± 0.4 50.0 ± 0.4 49.5 ± 0.4 48.7 ± 0.5 48.9 ± 0.4

Hemoglobin (g/dL) 

Day 3 13.7 ± 0.2 13.8 ± 0.2 13.5 ± 0.1 13.8 ± 0.1 13.8 ± 0.2

Day 21 15.4 ± 0.1 15.6 ± 0.1 15.5 ± 0.1 15.6 ± 0.1 15.4 ± 0.2

Week 13 15.8 ± 0.1 15.9 ± 0.1 16.0 ± 0.1 15.8 ± 0.2 15.8 ± 0.1

Erythrocytes (106

/μL) 

Day 3 6.27 ± 0.07 6.33 ± 0.09 6.18 ± 0.06 6.28 ± 0.05 6.36 ± 0.12

Day 21 7.25 ± 0.08 7.31 ± 0.06 7.34 ± 0.05 7.28 ± 0.08 7.09 ± 0.10

Week 13 8.96 ± 0.07 9.08 ± 0.06 9.03 ± 0.09 8.71 ± 0.09 8.67 ± 0.06*

Reticulocytes (106

/μL) 

Day 3 0.53 ± 0.03 0.58 ± 0.02 0.54 ± 0.04 0.50 ± 0.02 0.55 ± 0.03

Day 21 0.24 ± 0.01 0.23 ± 0.01 0.20 ± 0.02* 0.20 ± 0.01** 0.21 ± 0.01**

Week 13 0.21 ± 0.01 0.24 ± 0.02 0.24 ± 0.01 0.22 ± 0.01 0.22 ± 0.01

Nucleated erythrocytes (103

/μL) 

Day 3 0.17 ± 0.05 0.13 ± 0.06 0.12 ± 0.05 0.16 ± 0.02 0.13 ± 0.03

Day 21 0.05 ± 0.03 0.04 ± 0.03 0.01 ± 0.01 0.06 ± 0.03 0.07 ± 0.03

Week 13 0.02 ± 0.02 0.04 ± 0.03 0.00 ± 0.00b 0.02 ± 0.02b 0.00 ± 0.00

Mean cell volume (fL) 

Day 3 61.2 ± 0.3 61.5 ± 0.3 61.3 ± 0.3 61.6 ± 0.2 61.4 ± 0.2

Day 21 61.0 ± 0.4 60.7 ± 0.4 60.0 ± 0.3 61.6 ± 0.4 61.3 ± 0.3

Week 13 52.2 ± 0.2 53.4 ± 0.1** 53.0 ± 0.2** 53.4 ± 0.2** 53.6 ± 0.1**

Mean cell hemoglobin (pg) 

Day 3 21.8 ± 0.1 21.8 ± 0.2 21.9 ± 0.1 22.0 ± 0.1 21.7 ± 0.1

Day 21 21.2 ± 0.2 21.3 ± 0.1 21.1 ± 0.1 21.4 ± 0.1 21.7 ± 0.1

Week 13 17.6 ± 0.1 17.5 ± 0.1 17.7 ± 0.1 18.1 ± 0.1** 18.2 ± 0.1**

Mean cell hemoglobin concentration (g/dL) 

Day 3 35.6 ± 0.1 35.5 ± 0.2 35.7 ± 0.2 35.7 ± 0.2 35.4 ± 0.2 

Day 21 34.8 ± 0.3 35.1 ± 0.2 35.2 ± 0.2 34.8 ± 0.3 35.4 ± 0.2

Week 13 33.7 ± 0.2 32.8 ± 0.2* 33.4 ± 0.3 33.9 ± 0.2 34.0 ± 0.2

Platelets (103

/μL) 

Day 3 1,091.1 ± 30.5 1,086.9 ± 25.3 1,080.6 ± 14.8 1,025.7 ± 11.2 1,069.5 ± 26.3

Day 21 793.1 ± 19.9 807.4 ± 8.6 820.8 ± 10.4 824.7 ± 20.0 838.7 ± 28.7

Week 13 649.8 ± 10.3 640.6 ± 9.7 646.3 ± 10.8 641.4 ± 6.6 653.7 ± 13.7

Leukocytes (103

/μL)

Day 3 8.09 ± 0.28 8.49 ± 0.24 7.70 ± 0.24 7.89 ± 0.22 8.91 ± 0.54

Day 21 11.03 ± 0.41 12.07 ± 0.30 10.78 ± 0.45 10.78 ± 0.25 10.90 ± 0.59

Week 13 11.96 ± 0.53 12.60 ± 0.61 11.90 ± 0.38 12.86 ± 0.60 11.67 ± 0.36
Benzyltrimethylammonium Chloride, NTP TOX 57 C-5

TABLE C2

Hematology and Clinical Chemistry Data for Rats in the 13-Week Gavage Study 

of Benzyltrimethylammonium Chloride

Vehicle Control 12.5 mg/kg 25 mg/kg 50 mg/kg 100 mg/kg

Male (continued)

n

Day 3 10 10 10 10 10

Day 21 10 10 10 10 10

Week 13 10 9 10 10 10

Hematology (continued)

Segmented neutrophils (103

/μL) 

Day 3 1.28 ± 0.21 1.14 ± 0.07 0.96 ± 0.11 0.10 ± 0.11 1.14 ± 0.08

Day 21 1.17 ± 0.07 1.37 ± 0.19 1.18 ± 0.14 1.35 ± 0.12 1.15 ± 0.14

Week 13 2.13 ± 0.17 1.59 ± 0.11* 1.56 ± 0.16*b 1.54 ± 0.20*b 1.25 ± 0.18**

Lymphocytes (103

/μL) 

Day 3 6.69 ± 0.32 7.18 ± 0.19 6.64 ± 0.27 6.72 ± 0.18 7.57 ± 0.49

Day 21 9.55 ± 0.43 10.16 ± 0.25 9.17 ± 0.41 9.01 ± 0.27 9.49 ± 0.47

Week 13 9.06 ± 0.43 10.35 ± 0.65 9.82 ± 0.42b

 10.29 ± 0.61b 9.97 ± 0.50

Atypical lymphocytes (103

/μL) 

Day 21 0.04 ± 0.03 0.06 ± 0.05 0.00 ± 0.00 0.03 ± 0.02 0.00 ± 0.00

Week 13 0.22 ± 0.09 0.12 ± 0.05 0.18 ± 0.06b 0.19 ± 0.09b 0.16 ± 0.07

Monocytes (103

/μL) 

Day 3 0.09 ± 0.04 0.14 ± 0.06 0.08 ± 0.02 0.14 ± 0.05 0.19 ± 0.04

Day 21 0.22 ± 0.07 0.44 ± 0.09 0.37 ± 0.09 0.36 ± 0.07 0.24 ± 0.10

Week 13 0.46 ± 0.12 0.35 ± 0.07 0.18 ± 0.05b 0.53 ± 0.10b

 0.21 ± 0.04

Eosinophils (103

/μL) 

Day 3 0.02 ± 0.01 0.02 ± 0.01 0.03 ± 0.02 0.02 ± 0.02 0.01 ± 0.01

Day 21 0.05 ± 0.04 0.02 ± 0.02 0.04 ± 0.02 0.04 ± 0.02 0.01 ± 0.01

Week 13 0.08 ± 0.03 0.16 ± 0.04 0.12 ± 0.04b 0.11 ± 0.04b 0.07 ± 0.03

Clinical Chemistry

Urea nitrogen (mg/dL)

Day 3 20.1 ± 0.6 19.3 ± 0.4 19.4 ± 0.3 19.8 ± 0.3 19.2 ± 0.3

Day 21 22.4 ± 1.5 21.4 ± 0.5 20.9 ± 0.3 23.4 ± 1.8 21.8 ± 0.5

Week 13 23.5 ± 0.5 24.2 ± 1.3 23.6 ± 0.6 22.6 ± 0.3 22.3 ± 0.6

Creatinine (mg/dL) 

Day 3 0.58 ± 0.01 0.59 ± 0.01 0.58 ± 0.01 0.56 ± 0.02 0.57 ± 0.02

Day 21 0.68 ± 0.01 0.67 ± 0.02 0.68 ± 0.01 0.67 ± 0.02 0.67 ± 0.02

Week 13 0.66 ± 0.02 0.69 ± 0.01 0.70 ± 0.02 0.68 ± 0.01 0.67 ± 0.02

Total protein (g/dL) 

Day 3 5.7 ± 0.1 5.8 ± 0.1 5.7 ± 0.1 5.6 ± 0.1 5.7 ± 0.1

Day 21 6.2 ± 0.1 6.2 ± 0.1 6.2 ± 0.1 6.2 ± 0.1 6.2 ± 0.0

Week 13 6.8 ± 0.1 6.8 ± 0.1 7.0 ± 0.1 6.7 ± 0.1 6.7 ± 0.1

Albumin (g/dL) 

Day 3 4.3 ± 0.0 4.4 ± 0.0 4.3 ± 0.1 4.2 ± 0.1 4.3 ± 0.1

Day 21 4.6 ± 0.1 4.5 ± 0.0 4.5 ± 0.0 4.6 ± 0.1 4.6 ± 0.1

Week 13 5.0 ± 0.0 5.0 ± 0.1 5.0 ± 0.0 4.9 ± 0.0 4.9 ± 0.0

Alanine aminotransferase (IU/L) 

Day 3 42 ± 1 41 ± 2 41 ± 2 43 ± 2 42 ± 1

Day 21 47 ± 1 50 ± 1 46 ± 1 47 ± 1 46 ± 1

Week 13 67 ± 4 60 ± 4 69 ± 5 67 ± 5 56 ± 2
C-6 Benzyltrimethylammonium Chloride, NTP TOX 57

TABLE C2

Hematology and Clinical Chemistry Data for Rats in the 13-Week Gavage Study 

of Benzyltrimethylammonium Chloride

Vehicle Control 12.5 mg/kg 25 mg/kg 50 mg/kg 100 mg/kg

Male (continued)

n

Day 3 10 10 10 10 10

Day 21 10 10 10 10 10

Week 13 10 9 10 10 10

Clinical Chemistry (continued)

Alkaline phosphatase (IU/L) 

Day 3 708 ± 12 737 ± 17 730 ± 12 722 ± 15 700 ± 17

Day 21 533 ± 9 562 ± 17 543 ± 8 532 ± 10 544 ± 10

Week 13 270 ± 6 271± 8 276 ± 5 245 ± 5* 227 ± 7**

Creatine kinase (IU/L) 

Day 3 443 ± 47 577 ± 64 529 ± 38 429 ± 35 463 ± 31

Day 21 432 ± 31 457 ± 34 399 ± 20 467 ± 47 443 ± 47

Week 13 226 ± 23 255 ± 20 244 ± 14 256 ± 21 256 ± 30

Sorbitol dehydrogenase (IU/L) 

Day 3 16 ± 1 14 ± 2 15 ± 1 16 ± 1 16 ± 1

Day 21 19 ± 1 19 ± 1 20 ± 1 17 ± 1 20 ± 1

Week 13 25 ± 2 22 ± 2 24 ± 1 22 ± 1 19 ± 1**

Serum cholinesterase (IU/L) 

Day 3 859.6 ± 15.2 851.2 ± 17.9 853.5 ± 15.0 846.4 ± 14.4 904.1 ± 19.4

Day 21 692.9 ± 13.4 719.5 ± 11.1 712.4 ± 11.8 745.0 ± 32.0 777.3 ± 18.7**

Week 13 741.9 ± 20.7 760.0 ± 13.4 792.0 ± 25.2 792.5 ± 23.7 753.6 ± 16.3

Bile acids (μmol/L) 

Day 3 28.1 ± 4.2 18.0 ± 1.3 17.6 ± 1.3 23.3 ± 2.3 20.9 ± 1.6

Day 21 28.4 ± 2.9 15.6 ± 1.1* 18.6 ± 2.3 22.0 ± 2.5 26.8 ± 3.6

Week 13 19.4 ± 5.6 16.3 ± 2.7 17.3 ± 3.7 13.7 ± 0.8 17.6 ± 1.7

Female

n

Day 3 10 10 10 10 10

Day 21 10 10 10 10 10

Week 13 10 10 9 10 8

Hematology

Automated hematocrit (%) 

Day 3 42.9 ± 0.4 41.7 ± 0.5 41.9 ± 0.5 40.9 ± 0.5* 42.7 ± 0.4

Day 21 45.0 ± 0.4 45.2 ± 0.6 46.1 ± 0.3 45.0 ± 0.3 45.7 ± 0.4

Week 13 45.2 ± 0.4 46.8 ± 0.3* 46.2 ± 0.4 46.4 ± 0.3 44.8 ± 0.3

Manual hematocrit (%) 

Day 3 43.9 ± 0.4 42.5 ± 0.4 43.0 ± 0.4 42.9 ± 0.4 43.9 ± 0.5

Day 21 48.6 ± 0.4 48.1 ± 0.9 49.4 ± 0.5 48.1 ± 0.6 49.7 ± 0.4

Week 13 46.1 ± 0.3 47.2 ± 0.4 46.7 ± 0.3 47.0 ± 0.4 45.8 ± 0.4

Hemoglobin (g/dL) 

Day 3 14.7 ± 0.1 14.2 ± 0.1 14.3 ± 0.2 14.1 ± 0.2* 14.7 ± 0.1

Day 21 15.7 ± 0.1 15.7 ± 0.2 16.0 ± 0.1 15.7 ± 0.1 16.0 ± 0.1

Week 13 15.3 ± 0.1 15.7 ± 0.1* 15.5 ± 0.1 15.5 ± 0.1 15.1 ± 0.1
Benzyltrimethylammonium Chloride, NTP TOX 57 C-7

TABLE C2

Hematology and Clinical Chemistry Data for Rats in the 13-Week Gavage Study 

of Benzyltrimethylammonium Chloride

Vehicle Control 12.5 mg/kg 25 mg/kg 50 mg/kg 100 mg/kg

Female (continued)

n

Day 3 10 10 10 10 10

Day 21 10 10 10 10 10

Week 13 10 10 9 10 8

Hematology (continued)

Erythrocytes (106

/μL) 

Day 3 6.97 ± 0.08 6.74 ± 0.06 6.78 ± 0.10 6.61 ± 0.10* 6.91 ± 0.07

Day 21 7.15 ± 0.07 7.19 ± 0.12 7.31 ± 0.05 7.10 ± 0.07 7.31 ± 0.06

Week 13 7.88 ± 0.07 8.11 ± 0.05 7.97 ± 0.07 8.00 ± 0.06 7.70 ± 0.05

Reticulocytes (106

/μL) 

Day 3 0.28 ± 0.02 0.27 ± 0.02 0.25 ± 0.01 0.28 ± 0.01 0.30 ± 0.02

Day 21 0.14 ± 0.01 0.12 ± 0.01 0.14 ± 0.02 0.15 ± 0.01 0.14 ± 0.01

Week 13 0.18 ± 0.01 0.18 ± 0.01 0.18 ± 0.01 0.18 ± 0.01 0.19 ± 0.01

Nucleated erythrocytes (103

/μL) 

Day 3 0.13 ± 0.04 0.05 ± 0.03 0.08 ± 0.04 0.12 ± 0.04 0.06 ± 0.02

Day 21 0.06 ± 0.02 0.00 ± 0.00* 0.01 ± 0.01 0.04 ± 0.02 0.01 ± 0.01

Week 13 0.04 ± 0.02 0.00 ± 0.00 0.01 ± 0.01 0.01 ± 0.01 0.01 ± 0.01

Mean cell volume (fL) 

Day 3 61.6 ± 0.1 61.8 ± 0.2 61.8 ± 0.3 61.9 ± 0.3 61.8 ± 0.3 

Day 21 63.0 ± 0.2 62.9 ± 0.3 63.0 ± 0.3 63.4 ± 0.3 62.6 ± 0.3 

Week 13 57.4 ± 0.1 57.7 ± 0.1 57.9 ± 0.1** 57.9 ± 0.1** 58.2 ± 0.2**

Mean cell hemoglobin (pg) 

Day 3 21.1 ± 0.1 21.1 ± 0.1 21.1 ± 0.1 21.4 ± 0.2 21.3 ± 0.1

Day 21 21.9 ± 0.1 21.9 ± 0.2 21.9 ± 0.1 22.1 ± 0.2 21.9 ± 0.1 

Week 13 19.4 ± 0.1 19.4 ± 0.1 19.4 ± 0.1 19.4 ± 0.1 19.6 ± 0.1

Mean cell hemoglobin concentration (g/dL) 

Day 3 34.2 ± 0.1 34.2 ± 0.3 34.1 ± 0.2 34.5 ± 0.2 34.4 ± 0.2 

Day 21 34.9 ± 0.1 34.8 ± 0.2 34.8 ± 0.1 34.9 ± 0.3 35.1 ± 0.2 

Week 13 33.9 ± 0.1 33.7 ± 0.2 33.5 ± 0.2 33.4 ± 0.2 33.6 ± 0.2

Platelets (103

/μL) 

Day 3 1,010.4 ± 33.0 1,056.0 ± 14.6 1,045.4 ± 20.1 1,016.0 ± 20.9 980.5 ± 24.8 

Day 21 693.6 ± 15.7 696.2 ± 17.9 709.2 ± 16.2 723.9 ± 20.5 665.5 ± 12.8 

Week 13 649.3 ± 13.8 653.2 ± 11.5 648.7 ± 20.6 708.8 ± 35.8 641.0 ± 16.4

Leukocytes (103

/μL)

Day 3 9.52 ± 0.28 9.39 ± 0.28 9.18 ± 0.26 9.24 ± 0.41 9.77 ± 0.33

Day 21 10.73 ± 0.48 10.20 ± 0.71 10.72 ± 0.58 9.71 ± 0.48 10.51 ± 0.36

Week 13 10.38 ± 0.32 9.85 ± 0.60 9.84 ± 0.42 10.71 ± 0.52 10.51 ± 0.63

Segmented neutrophils (103

/μL)

Day 3 1.08 ± 0.04 1.21 ± 0.11 1.23 ± 0.12 1.21 ± 0.08 1.10 ± 0.15

Day 21 1.06 ± 0.09 1.19 ± 0.15 1.02 ± 0.07 0.98 ± 0.11 1.06 ± 0.09

Week 13 1.30 ± 0.17 0.85 ± 0.11 1.72 ± 0.23 1.13 ± 0.18 1.05 ± 0.20

Lymphocytes (103

/μL) 

Day 3 8.11 ± 0.29 7.71 ± 0.24 7.51 ± 0.29 7.53 ± 0.41 8.26 ± 0.23

Day 21 9.42 ± 0.45 8.79 ± 0.58 9.46 ± 0.61 8.57 ± 0.48 9.33 ± 0.41

Week 13 8.53 ± 0.34 8.45 ± 0.50 7.48 ± 0.41 8.84 ± 0.38 8.93 ± 0.61

Atypical lymphocytes (103

/μL) 

Day 3 0.01 ± 0.01 0.03 ± 0.02 0.01 ± 0.01 0.01 ± 0.01 0.00 ± 0.00

Week 13 0.02 ± 0.02 0.24 ± 0.20 0.08 ± 0.04 0.13 ± 0.05 0.11 ± 0.04
C-8 Benzyltrimethylammonium Chloride, NTP TOX 57

TABLE C2

Hematology and Clinical Chemistry Data for Rats in the 13-Week Gavage Study 

of Benzyltrimethylammonium Chloride

Vehicle Control 12.5 mg/kg 25 mg/kg 50 mg/kg 100 mg/kg

Female (continued)

n

Day 3 10 10 10 10 10

Day 21 10 10 10 10 10

Week 13 10 10 9 10 8

Hematology (continued)

Monocytes (103

/μL)

Day 3 0.29 ± 0.09 0.42 ± 0.09 0.36 ± 0.09 0.38 ± 0.10 0.35 ± 0.09

Day 21 0.09 ± 0.05 0.09 ± 0.05 0.11 ± 0.05 0.12 ± 0.06 0.05 ± 0.03

Week 13 0.35 ± 0.07 0.37 ± 0.08 0.40 ± 0.07 0.49 ± 0.05 0.39 ± 0.11

Eosinophils (103

/μL) 

Day 3 0.02 ± 0.01 0.01 ± 0.01 0.05 ± 0.02 0.03 ± 0.01 0.04 ± 0.02

Day 21 0.07 ± 0.02 0.06 ± 0.03 0.06 ± 0.02 0.01 ± 0.01* 0.00 ± 0.00**

Week 13 0.17 ± 0.02 0.15 ± 0.05 0.14 ± 0.04 0.10 ± 0.02 0.01 ± 0.01**

Clinical Chemistry

Urea nitrogen (mg/dL) 

Day 3 21.7 ± 0.6 20.8 ± 0.7 19.6 ± 0.5 21.5 ± 0.5 22.6 ± 0.9

Day 21 22.8 ± 0.7 22.7 ± 0.6 21.7 ± 0.7 24.8 ± 1.4 23.4 ± 0.7

Week 13 21.2 ± 0.6 24.2 ± 0.8* 21.4 ± 0.8 21.8 ± 1.3 22.8 ± 0.7

Creatinine (mg/dL) 

Day 3 0.57 ± 0.02 0.57 ± 0.02 0.57 ± 0.02 0.58 ± 0.01 0.58 ± 0.01

Day 21 0.60 ± 0.00 0.65 ± 0.02 0.65 ± 0.02 0.64 ± 0.02 0.62 ± 0.01

Week 13 0.69 ± 0.02 0.69 ± 0.01 0.68 ± 0.02 0.69 ± 0.02 0.65 ± 0.03

Total protein (g/dL) 

Day 3 5.9 ± 0.1 5.8 ± 0.1 5.6 ± 0.1* 5.6 ± 0.0* 5.7 ± 0.1

Day 21 5.8 ± 0.0 5.8 ± 0.0 5.8 ± 0.1 5.8 ± 0.1 5.7 ± 0.1

Week 13 6.8 ± 0.1 6.7 ± 0.1 6.5 ± 0.1* 6.4 ± 0.1** 6.4 ± 0.1**

Albumin (g/dL) 

Day 3 4.5 ± 0.0 4.4 ± 0.1 4.3 ± 0.1 4.3 ± 0.1* 4.3 ± 0.0**

Day 21 4.5 ± 0.0 4.4 ± 0.0 4.5 ± 0.1 4.4 ± 0.0 4.4 ± 0.1

Week 13 5.0 ± 0.1 5.0 ± 0.0 4.9 ± 0.1 4.7 ± 0.1** 4.6 ± 0.1** 

Alanine aminotransferase (IU/L) 

Day 3 37 ± 1 36 ± 1 36 ± 1 36 ± 2 36 ± 1

Day 21 36 ± 1 34 ± 1 34 ± 1 39 ± 1 39 ± 1

Week 13 42 ± 3 51 ± 4 41 ± 2 43 ± 2 48 ± 3

Alkaline phosphatase (IU/L)

Day 3 541 ± 13 540 ± 13 537 ± 9 544 ± 13 544 ± 15

Day 21 398 ± 9 382 ± 8 400 ± 8 405 ± 13 383 ± 12

Week 13 212 ± 7 237 ± 5 235 ± 9 208 ± 6 233 ± 8

Creatine kinase (IU/L) 

Day 3 517 ± 44 574 ± 60 488 ± 44 484 ± 41 474 ± 33

Day 21 493 ± 60 505 ± 48 502 ± 29 387 ± 42 405 ± 46

Week 13 366 ± 29 311 ± 33 395 ± 25 309 ± 47 392 ± 44

Sorbitol dehydrogenase (IU/L) 

Day 3 13 ± 1 14 ± 2 13 ± 1 14 ± 1 14 ± 1

Day 21 18 ± 1 18 ± 2 19 ± 1 21 ± 1 18 ± 1

Week 13 17 ± 1 20 ± 1 15 ± 1 16 ± 2 13 ± 1
Benzyltrimethylammonium Chloride, NTP TOX 57 C-9

TABLE C2

Hematology and Clinical Chemistry Data for Rats in the 13-Week Gavage Study 

of Benzyltrimethylammonium Chloride

Vehicle Control 12.5 mg/kg 25 mg/kg 50 mg/kg 100 mg/kg

Female (continued)

n

Day 3 10 10 10 10 10

Day 21 10 10 10 10 10

Week 13 10 10 9 10 8

Clinical Chemistry (continued)

Serum cholinesterase (IU/L) 

Day 3 1,748.3 ± 49.9 1,774.0 ± 72.4 1,701.2 ± 94.9 1,764.3 ± 95.6 1,895.4 ± 76.1

Day 21 2,525.4 ± 68.3 2,872.1 ± 127.7 2,865.9 ± 143.1 2,819.0 ± 167.2 2,781.5 ± 181.6

Week 13 4,297.5 ± 144.8 4,412.9 ± 154.0 4,034.1 ± 206.4 3,284.1 ± 134.2** 2,797.9 ± 246.9**

Bile acids (μmol/L) 

Day 3 24.1 ± 2.3 20.8 ± 2.8 17.1 ± 1.5 21.8 ± 3.2 18.1 ± 2.6

Day 21 25.9 ± 1.5 20.9 ± 1.6 18.1 ± 2.6* 28.9 ± 4.7 20.4 ± 3.4

Week 13 14.9 ± 0.9 21.8 ± 4.0 20.1 ± 2.8 35.0 ± 8.6** 27.8 ± 3.9*

* Significantly different (P#0.05) from the vehicle control group by Dunnís or Shirleyís test

** P#0.01 a Mean ± standard error. Statistical tests were performed on unrounded data. b n=9
C-10 Benzyltrimethylammonium Chloride, NTP TOX 57

TABLE C3

Hematology and Clinical Chemistry Data for Mice in the 16-Day Gavage Study 

of Benzyltrimethylammonium Chloridea

Vehicle Control 63 mg/kg 125 mg/kg

Male

n 5 5 5

Hematology

Automated hematocrit (%) 45.0 ± 0.3 45.5 ± 0.5 45.5 ± 0.7 

Manual hematocrit (%) 50.2 ± 0.4 51.4 ± 0.4 50.4 ± 0.5 

Hemoglobin (g/dL) 15.8 ± 0.2 15.7 ± 0.2 15.9 ± 0.2

Erythrocytes (106

/μL) 9.00 ± 0.08 9.09 ± 0.11 9.10 ± 0.12

Reticulocytes (106

/μL) 0.19 ± 0.03 0.15 ± 0.02 0.18 ± 0.02

Nucleated erythrocytes/100 leukocytes 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00

Mean cell volume (fL) 50.0 ± 0.2 50.0 ± 0.2 50.0 ± 0.1

Mean cell hemoglobin (pg) 17.5 ± 0.0 17.3 ± 0.1 17.5 ± 0.1

Mean cell hemoglobin

 concentration (g/dL) 35.0 ± 0.1 34.6 ± 0.4 34.9 ± 0.3 

Platelets (103

/μL) 760.0 ± 24.3 718.6 ± 36.0 763.2 ± 38.4

Leukocytes (103

/μL) 4.84 ± 0.39 4.98 ± 0.56 4.98 ± 0.54 

Segmented neutrophils (103

/μL) 0.53 ± 0.13 0.42 ± 0.06 0.59 ± 0.09

Lymphocytes (103

/μL) 4.20 ± 0.27 4.44 ± 0.53 4.26 ± 0.44 

Monocytes (103

/μL) 0.08 ± 0.04 0.09 ± 0.02 0.05 ± 0.02 

Eosinophils (103

/μL) 0.03 ± 0.02 0.04 ± 0.02 0.08 ± 0.03

Clinical Chemistry

Serum cholinesterase (IU/L) 5,620.2 ± 192.7 5,900.0 ± 160.0 5,941.8 ± 199.0 

Erythrocyte cholinesterase (IU/L) 2,016.0 ± 185.2 1,891.4 ± 247.4 1,812.5 ± 82.1b
Benzyltrimethylammonium Chloride, NTP TOX 57 C-11

TABLE C3

Hematology and Clinical Chemistry Data for Mice in the 16-Day Gavage Study 

of Benzyltrimethylammonium Chloride

Vehicle Control 63 mg/kg 125 mg/kg

Female

n 5 5 4

Hematology

Automated hematocrit (%) 44.1 ± 0.5 43.4 ± 0.5 43.9 ± 0.5

Manual hematocrit (%) 50.0 ± 0.4 49.4 ± 0.6 50.0 ± 0.9

Hemoglobin (g/dL) 15.6 ± 0.3 15.5 ± 0.2 15.4 ± 0.2

Erythrocytes (106

/μL) 8.92 ± 0.09 8.75 ± 0.09 8.81 ± 0.14 

Reticulocytes (106

/μL) 0.22 ± 0.04 0.20 ± 0.06 0.16 ± 0.03

Nucleated erythrocytes/100 leukocytes 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00c

Mean cell volume (fL) 49.4 ± 0.1 49.6 ± 0.2 49.9 ± 0.3 

Mean cell hemoglobin (pg) 17.5 ± 0.1 17.7 ± 0.1 17.5 ± 0.1 

Mean cell hemoglobin 

 concentration (g/dL) 35.3 ± 0.3 35.6 ± 0.2 35.0 ± 0.1

Platelets (103

/μL) 708.2 ± 21.8 738.6 ± 12.6 722.3 ± 22.9

Leukocytes (103

/μL) 4.58 ± 0.48 5.26 ± 0.38 5.93 ± 0.49 

Segmented neutrophils (103

/μL) 0.64 ± 0.16 0.58 ± 0.09 0.51 ± 0.12 

Lymphocytes (103

/μL) 3.86 ± 0.46 4.50 ± 0.34 5.36 ± 0.37 

Monocytes (103

/μL) 0.05 ± 0.02 0.10 ± 0.06 0.07 ± 0.03 

Eosinophils (103

/μL) 0.03 ± 0.02 0.07 ± 0.02 0.00 ± 0.00 

Clinical Chemistry

Serum cholinesterase (IU/L) 8,155.8 ± 24.2 8,328.6 ± 186.0 8,038.5 ± 247.4

Erythrocyte cholinesterase (IU/L) 2,044.8 ± 443.0 2,888.2 ± 300.2 2,384.8 ± 560.3

a Mean ± standard error. Statistical tests were performed on unrounded data. No data are available for the 250, 500, and 1,000 mg/kg groups

due to 100% mortality. b n=4 c n=5
C-12 Benzyltrimethylammonium Chloride, NTP TOX 57

TABLE C4

Clinical Chemistry Data for Mice in the 13-Week Gavage Study of Benzyltrimethylammonium Chloridea

Vehicle Control 12.5 mg/kg 25 mg/kg 50 mg/kg 100 mg/kg

Male

n 8 10 10 10 9

Urea nitrogen (mg/dL) 27.0 ± 0.6b 31.6 ± 1.6 30.3 ± 1.8 30.8 ± 1.1 30.1 ± 1.7c

Creatinine (mg/dL) 0.43 ± 0.02 0.48 ± 0.02 0.46 ± 0.02 0.47 ± 0.02 0.44 ± 0.02c

Total protein (g/dL) 6.1 ± 0.1d 6.0 ± 0.1b 5.8 ± 0.1** 5.7 ± 0.1**b 5.7 ± 0.1**e

Albumin (g/dL) 4.3 ± 0.1 4.3 ± 0.1 4.1 ± 0.1 4.2 ± 0.1 4.1 ± 0.1c

Alanine aminotransferase (IU/L) 40 ± 8 53 ± 20 84 ± 31 71 ± 22 37 ± 8

Alkaline phosphatase ((IU/L) 91 ± 2b 86 ± 4 86 ± 2 86 ± 3 84 ± 3

Creatine kinase (IU/L) 276 ± 87b 255 ± 57 461 ± 113b 485 ± 135 310 ± 77

Sorbitol dehydrogenase (IU/L) 47 ± 2 46 ± 1 45 ± 2 45 ± 4 45 ± 1c

Serum cholinesterase (IU/L) 8,963 ± 210 8,923 ± 376 8,404 ± 107 8,380 ± 353*b 8,893 ± 369c

Bile acids (μmol/L) 14.0 ± 1.1 13.8 ± 0.6b 14.9 ± 0.8 15.2 ± 0.8b 13.3 ± 0.6e

Female

n 10 10 10 10 9

Urea nitrogen (mg/dL) 26.0 ± 2.0 26.9 ± 1.2 28.9 ± 1.3 25.0 ± 1.8 26.3 ± 1.3

Creatinine (mg/dL) 0.59 ± 0.02 0.58 ± 0.02 0.60 ± 0.02 0.54 ± 0.02 0.58 ± 0.02 

Total protein (g/dL) 6.0 ± 0.1 5.9 ± 0.1 5.9 ± 0.1 5.8 ± 0.1 5.8 ± 0.1

Albumin (g/dL) 4.6 ± 0.1 4.6 ± 0.1 4.5 ± 0.1 4.4 ± 0.0 4.4 ± 0.1

Alanine aminotransferase (IU/L) 34 ± 6 40 ± 9 29 ± 2 26 ± 1 36 ± 9

Alkaline phosphatase ((IU/L) 139 ± 3 126 ± 6 124 ± 4 128 ± 6 125 ± 6

Creatine kinase (IU/L) 297 ± 69 615 ± 212 428 ± 55 474 ± 91 318 ± 76

Sorbitol dehydrogenase (IU/L) 47 ± 1 48 ± 3 48 ± 1 48 ± 1 46 ± 1

Serum cholinesterase (IU/L) 10,020 ± 235 10,147 ± 114 9,964 ± 179 10,080 ± 230 9,726 ± 146

Bile acids (μmol/L) 15.1 ± 0.6 15.5 ± 0.8 15.5 ± 0.5 15.2 ± 0.5 15.6 ± 0.2

* Significantly different (P#0.05) from the vehicle control group by Dunnís test

** Significantly different (P#0.01) from the vehicle control group by Shirleyís test a Mean ± standard error. Statistical tests were performed on unrounded data. b n=9 c n=8

d n=5 e n=7
D-1

APPENDIX D

ORGAN WEIGHTS AND

ORGAN-WEIGHT-TO-BODY-WEIGHT RATIOS 

TABLE D1 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats 

in the 16-Day Gavage Study of Benzyltrimethylammonium Chloride . . . . . . . . . . . . . D-2

TABLE D2 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats

in the 13-Week Gavage Study of Benzyltrimethylammonium Chloride . . . . . . . . . . . . D-3

TABLE D3 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Mice 

in the 16-Day Gavage Study of Benzyltrimethylammonium Chloride . . . . . . . . . . . . . D-4

TABLE D4 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Mice 

in the 13-Week Gavage Study of Benzyltrimethylammonium Chloride . . . . . . . . . . . . D-5
D-2 Benzyltrimethylammonium Chloride, NTP TOX 57

TABLE D1

Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats in the 16-Day Gavage Study

of Benzyltrimethylammonium Chloridea

Vehicle Control 16 mg/kg 32 mg/kg 63 mg/kg

n 5 5 5 5

Male

Necropsy body wt 240 ± 4 231 ± 5 233 ± 12 226 ± 8

Heart

Absolute 0.833 ± 0.013 0.810 ± 0.016 0.828 ± 0.046 0.829 ± 0.038

Relative 3.46 ± 0.05 3.51 ± 0.06 3.55 ± 0.04 3.67 ± 0.11

R. Kidney 

Absolute 1.078 ± 0.024 1.061 ± 0.029 1.058 ± 0.081 1.011 ± 0.020

Relative 4.49 ± 0.10 4.59 ± 0.09 4.51 ± 0.14 4.48 ± 0.08

Liver

Absolute 12.166 ± 0.302 11.288 ± 0.400 11.509 ± 0.970 10.997 ± 0.626

Relative 50.58 ± 0.70 48.82 ± 0.78 48.98 ± 1.68 48.47 ± 1.20

Lung

Absolute 1.383 ± 0.058 1.355 ± 0.087 1.491 ± 0.104 1.314 ± 0.053

Relative 5.75 ± 0.20 5.85 ± 0.27 6.39 ± 0.30 5.82 ± 0.21

Spleen

Absolute 0.626 ± 0.017 0.585 ± 0.020 0.605 ± 0.029 0.579 ± 0.024

Relative 2.60 ± 0.05 2.53 ± 0.05 2.60 ± 0.04 2.56 ± 0.04

R. Testis

Absolute 1.266 ± 0.013 1.247 ± 0.026 1.231 ± 0.055 1.231 ± 0.019

Relative 5.27 ± 0.09 5.41 ± 0.14 5.28 ± 0.06 5.46 ± 0.11

Thymus

Absolute 0.521 ± 0.010 0.501 ± 0.010 0.523 ± 0.029 0.502 ± 0.035

Relative 2.17 ± 0.06 2.17 ± 0.05 2.26 ± 0.13 2.21 ± 0.09

Female

Necropsy body wt 137 ± 5 144 ± 5 142 ± 4 137 ± 4

Heart

Absolute 0.548 ± 0.020 0.577 ± 0.024 0.596 ± 0.009 0.560 ± 0.021

Relative 4.01 ± 0.05 4.02 ± 0.05 4.21 ± 0.08 4.10 ± 0.12

R. Kidney

Absolute 0.636 ± 0.031 0.655 ± 0.023 0.663 ± 0.020 0.619 ± 0.025

Relative 4.65 ± 0.12 4.57 ± 0.05 4.67 ± 0.11 4.53 ± 0.11

Liver

Absolute 5.966 ± 0.333 6.233 ± 0.278 6.147 ± 0.219 5.771 ± 0.324

Relative 43.58 ± 1.29 43.37 ± 0.80 43.30 ± 1.00 42.09 ± 1.13

Lung

Absolute 0.897 ± 0.041 0.985 ± 0.043 0.944 ± 0.034 0.884 ± 0.063

Relative 6.56 ± 0.13 6.88 ± 0.29 6.65 ± 0.18 6.44 ± 0.27

Spleen

Absolute 0.401 ± 0.013 0.416 ± 0.017 0.426 ± 0.016 0.366 ± 0.021

Relative 2.93 ± 0.02 2.90 ± 0.08 3.00 ± 0.11 2.67 ± 0.07

Thymus

Absolute 0.357 ± 0.022 0.367 ± 0.012 0.366 ± 0.008 0.353 ± 0.023

Relative 2.61 ± 0.13 2.56 ± 0.08 2.58 ± 0.09 2.58 ± 0.11

a Organ weights (absolute weights) and body weights are given in grams; organ-weight-to-body-weight ratios (relative weights) are given as

mg organ weight/g body weight (mean ± standard error). Differences from the vehicle control group were not significant by Dunnettís test.

No data are available for the 125 and 250 mg/kg groups due to 100% mortality.
Benzyltrimethylammonium Chloride, NTP TOX 57 D-3

TABLE D2

Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats in the 13-Week Gavage Study

of Benzyltrimethylammonium Chloridea

Vehicle Control 12.5 mg/kg 25 mg/kg 50 mg/kg 100 mg/kg

Male

n 10 10 10 10 10

Necropsy body wt 338 ± 8 337 ± 8 336 ± 8 340 ± 5 311 ± 9 

Heart

Absolute 1.018 ± 0.030 1.010 ± 0.024 1.000 ± 0.024 1.016 ± 0.025 0.953 ± 0.028

Relative 3.02 ± 0.05 3.00 ± 0.04 2.98 ± 0.05 2.99 ± 0.06 3.07 ± 0.03

R. Kidney

Absolute 1.266 ± 0.049 1.274 ± 0.044 1.307 ± 0.092 1.238 ± 0.036 1.208 ± 0.041

Relative 3.74 ± 0.07 3.78 ± 0.07 3.89 ± 0.24 3.64 ± 0.08 3.89 ± 0.05 

Liver

Absolute 12.136 ± 0.423 12.671 ± 0.412 11.893 ± 0.277 12.146 ± 0.262 11.409 ± 0.459 

Relative 35.88 ± 0.50 37.58 ± 0.69 35.46 ± 0.34 35.78 ± 0.60 36.66 ± 0.73 

Lung

Absolute 1.540 ± 0.040 1.473 ± 0.045 1.503 ± 0.045 1.515 ± 0.042 1.388 ± 0.054 

Relative 4.57 ± 0.08 4.37 ± 0.07 4.48 ± 0.09 4.46 ± 0.09 4.47 ± 0.10 

R. Testis

Absolute 1.459 ± 0.043 1.380 ± 0.036 1.385 ± 0.063 1.443 ± 0.029 1.423 ± 0.043 

Relative 4.32 ± 0.07 4.11 ± 0.12 4.15 ± 0.21 4.25 ± 0.05 4.58 ± 0.02 

Thymus

Absolute 0.328 ± 0.016 0.343 ± 0.023 0.341 ± 0.022 0.360 ± 0.017 0.295 ± 0.012 

Relative 0.97 ± 0.04 1.01 ± 0.05 1.02 ± 0.06 1.06 ± 0.05 0.96 ± 0.04 

Female

n 10 10 9 9 8

Necropsy body wt 190 ± 3 198 ± 4 193 ± 3 193 ± 4 187 ± 4 

Heart

Absolute 0.704 ± 0.011 0.679 ± 0.012 0.674 ± 0.016 0.693 ± 0.013 0.670 ± 0.014 

Relative 3.71 ± 0.04 3.43 ± 0.05** 3.49 ± 0.05* 3.60 ± 0.07 3.59 ± 0.06 

R. Kidney

Absolute 0.716 ± 0.015 0.730 ± 0.020 0.710 ± 0.012 0.750 ± 0.014 0.730 ± 0.025 

Relative 3.77 ± 0.07 3.68 ± 0.06 3.68 ± 0.05 3.90 ± 0.06 3.91 ± 0.10 

Liver

Absolute 6.435 ± 0.144 6.532 ± 0.221 6.449 ± 0.129 6.715 ± 0.204 6.731 ± 0.221 

Relative 33.82 ± 0.32 32.92 ± 0.81 33.47 ± 0.54 34.79 ± 0.54 36.00 ± 0.68* 

Lung

Absolute 1.156 ± 0.048 1.184 ± 0.033 1.147 ± 0.037 1.195 ± 0.050 1.121 ± 0.045 

Relative 6.06 ± 0.17 5.97 ± 0.13 5.97 ± 0.24 6.20 ± 0.24 6.00 ± 0.17 

Thymus

Absolute 0.263 ± 0.010 0.276 ± 0.014 0.263 ± 0.010 0.251 ± 0.013 0.257 ± 0.012 

Relative 1.38 ± 0.04 1.39 ± 0.06 1.36 ± 0.04 1.30 ± 0.05 1.37 ± 0.06 

* Significantly different (P#0.05) from the vehicle control group by Williamsí or Dunnettís test

** Significantly different (P#0.01) from the vehicle control group by Dunnettís test a Organ weights (absolute weights) and body weights are given in grams; organ-weight-to-body-weight ratios (relative weights) are given as

mg organ weight/g body weight (mean ± standard error).
D-4 Benzyltrimethylammonium Chloride, NTP TOX 57

TABLE D3

Organ Weights and Organ-Weight-to-Body-Weight Ratios for Mice in the 16-Day Gavage Study

of Benzyltrimethylammonium Chloridea

Vehicle Control 63 mg/kg 125 mg/kg

Male

n 5 5 5

Necropsy body wt 26.4 ± 0.7 26.0 ± 0.5 26.1 ± 0.6

Heart

Absolute 0.132 ± 0.005 0.130 ± 0.003 0.131 ± 0.003

Relative 5.00 ± 0.11 5.00 ± 0.06 5.01 ± 0.05

R. Kidney

Absolute 0.249 ± 0.012 0.246 ± 0.007 0.255 ± 0.008

Relative 9.41 ± 0.22 9.43 ± 0.13 9.76 ± 0.11

Liver

Absolute 1.456 ± 0.049 1.416 ± 0.044 1.437 ± 0.042

Relative 55.03 ± 0.66 54.35 ± 1.03 54.94 ± 0.64

Lung

Absolute 0.180 ± 0.007 0.184 ± 0.007 0.194 ± 0.006

Relative 6.80 ± 0.20 7.08 ± 0.35 7.42 ± 0.18

Spleen

Absolute 0.069 ± 0.003 0.067 ± 0.003 0.067 ± 0.003

Relative 2.61 ± 0.06 2.57 ± 0.08 2.57 ± 0.07

R. Testis

Absolute 0.106 ± 0.002 0.103 ± 0.001 0.103 ± 0.004

Relative 4.03 ± 0.16 3.96 ± 0.07 3.94 ± 0.14

Thymus

Absolute 0.054 ± 0.003 0.049 ± 0.004 0.051 ± 0.005

Relative 2.04 ± 0.13 1.87 ± 0.16 1.96 ± 0.20

Female 

n 5 5 4

Necropsy body wt 21.2 ± 0.4 21.9 ± 0.5 22.4 ± 0.1

Heart

Absolute 0.120 ± 0.003 0.118 ± 0.005 0.118 ± 0.002 

Relative 5.66 ± 0.11 5.40 ± 0.14 5.28 ± 0.09 

R. Kidney

Absolute 0.169 ± 0.007 0.173 ± 0.002 0.173 ± 0.007 

Relative 7.96 ± 0.22 7.90 ± 0.12 7.75 ± 0.32 

Liver

Absolute 1.073 ± 0.043 1.136 ± 0.025 1.209 ± 0.017*

Relative 50.51 ± 1.35 51.88 ± 0.69 54.12 ± 1.04 

Lung

Absolute 0.171 ± 0.007 0.162 ± 0.008 0.150 ± 0.009

Relative 8.07 ± 0.23 7.38 ± 0.25 6.72 ± 0.36* 

Spleen

Absolute 0.078 ± 0.003 0.078 ± 0.004 0.076 ± 0.004

Relative 3.67 ± 0.12 3.56 ± 0.09 3.40 ± 0.20 

Thymus

Absolute 0.062 ± 0.003 0.064 ± 0.005 0.064 ± 0.007 

Relative 2.94 ± 0.14 2.91 ± 0.21 2.84 ± 0.29 

* Significantly different (P#0.05) from the vehicle control group by Williamsí or Dunnettís test a Organ weights (absolute weights) and body weights are given in grams; organ-weight-to-body-weight ratios (relative weights) are given as

mg organ weight/g body weight (mean ± standard error). No data are available for the 250, 500, and 1,000 mg/kg groups due to 100%

mortality.
Benzyltrimethylammonium Chloride, NTP TOX 57 D-5

TABLE D4

Organ Weights and Organ-Weight-to-Body-Weight Ratios for Mice in the 13-Week Gavage Study

of Benzyltrimethylammonium Chloridea

Vehicle Control 12.5 mg/kg 25 mg/kg 50 mg/kg 100 mg/kg

Male

n 10 10 10 8 9

Necropsy body wt 34.9 ± 0.7 34.9 ± 1.0 34.5 ± 0.6 34.8 ± 0.9 33.9 ± 0.9 

Heart

Absolute 0.142 ± 0.003 0.146 ± 0.003 0.146 ± 0.002 0.151 ± 0.003 0.145 ± 0.003 

Relative 4.06 ± 0.06 4.19 ± 0.06 4.25 ± 0.05* 4.30 ± 0.10* 4.29 ± 0.06* 

R. Kidney

Absolute 0.284 ± 0.006 0.290 ± 0.008 0.285 ± 0.006 0.311 ± 0.008* 0.297 ± 0.009 

Relative 8.13 ± 0.13 8.32 ± 0.17 8.28 ± 0.16 8.82 ± 0.15** 8.77 ± 0.16** 

Liver

Absolute 1.519 ± 0.042 1.553 ± 0.049 1.534 ± 0.049 1.599 ± 0.041 1.537 ± 0.036 

Relative 43.44 ± 0.61 44.48 ± 0.57 44.47 ± 0.90 45.33 ± 0.50 45.39 ± 1.00 

Lung

Absolute 0.196 ± 0.011 0.199 ± 0.008 0.198 ± 0.014 0.188 ± 0.015 0.184 ± 0.006 

Relative 5.63 ± 0.32 5.71 ± 0.17 5.78 ± 0.46 5.31 ± 0.34 5.43 ± 0.18 

R. Testis

Absolute 0.119 ± 0.005 0.113 ± 0.007 0.119 ± 0.003 0.120 ± 0.005 0.120 ± 0.004 

Relative 3.39 ± 0.11 3.24 ± 0.21 3.46 ± 0.07 3.39 ± 0.12 3.54 ± 0.05 

Thymus

Absolute 0.063 ± 0.009 0.045 ± 0.006 0.044 ± 0.004 0.042 ± 0.005 0.044 ± 0.005 

Relative 1.80 ± 0.25 1.31 ± 0.19 1.26 ± 0.11 1.19 ± 0.14 1.31 ± 0.14 

Female

n 10 10 10 10 9

Necropsy body wt 29.1 ± 1.0 29.9 ± 0.9 28.7 ± 0.9 29.2 ± 1.3 28.2 ± 0.9 

Heart

Absolute 0.125 ± 0.002 0.128 ± 0.003 0.122 ± 0.003 0.126 ± 0.003 0.124 ± 0.003 

Relative 4.31 ± 0.12 4.31 ± 0.09 4.28 ± 0.11 4.37 ± 0.12 4.40 ± 0.08 

R. Kidney

Absolute 0.193 ± 0.003 0.190 ± 0.004 0.184 ± 0.004 0.192 ± 0.006 0.184 ± 0.006 

Relative 6.70 ± 0.22 6.40 ± 0.18 6.44 ± 0.14 6.64 ± 0.17 6.52 ± 0.06 

Liver

Absolute 1.168 ± 0.022 1.223 ± 0.046 1.246 ± 0.034 1.249 ± 0.055 1.208 ± 0.039 

Relative 40.47 ± 1.35 40.95 ± 0.81 43.57 ± 0.50 42.95 ± 1.21 42.81 ± 0.64 

Lung

Absolute 0.187 ± 0.004 0.196 ± 0.009 0.172 ± 0.005 0.176 ± 0.006 0.176 ± 0.006 

Relative 6.48 ± 0.24 6.58 ± 0.27 6.02 ± 0.12 6.08 ± 0.16 6.25 ± 0.14 

Thymus

Absolute 0.051 ± 0.003 0.054 ± 0.003 0.048 ± 0.002 0.056 ± 0.005 0.050 ± 0.004 

Relative 1.77 ± 0.09 1.79 ± 0.09 1.69 ± 0.10 1.90 ± 0.12 1.78 ± 0.16 

* Significantly different (P#0.05) from the vehicle control group by Williamsí or Dunnettís test

** Significantly different (P#0.01) from the vehicle control group by Williamsí test a Organ weights (absolute weights) and body weights are given in grams; organ-weight-to-body-weight ratios (relative weights) are given as

mg organ weight/g body weight (mean ± standard error).
D-6 Benzyltrimethylammonium Chloride, NTP TOX 57
E-1

APPENDIX E

REPRODUCTIVE TISSUE EVALUATIONS

AND ESTROUS CYCLE CHARACTERIZATION

TABLE E1 Summary of Reproductive Tissue Evaluations for Male Rats 

in the 13-Week Gavage Study of Benzyltrimethylammonium Chloride . . . . . . . . . . . . E-2

TABLE E2 Estrous Cycle Characterization for Female Rats 

in the 13-Week Gavage Study of Benzyltrimethylammonium Chloride . . . . . . . . . . . . E-2

TABLE E3 Summary of Reproductive Tissue Evaluations for Male Mice 

in the 13-Week Gavage Study of Benzyltrimethylammonium Chloride . . . . . . . . . . . . E-3

TABLE E4 Estrous Cycle Characterization for Female Mice

in the 13-Week Gavage Study of Benzyltrimethylammonium Chloride . . . . . . . . . . . . E-3
E-2 Benzyltrimethylammonium Chloride, NTP TOX 57

TABLE E1

Summary of Reproductive Tissue Evaluations for Male Rats in the 13-Week Gavage Study

of Benzyltrimethylammonium Chloridea

Vehicle Control 25 mg/kg 50 mg/kg 100 mg/kg

n 10 10 10 10

Weights (g)

Necropsy body wt 338 ± 9 335 ± 8 340 ± 5 311 ± 9*

L. Cauda epididymis 0.1660 ± 0.0076 0.1633 ± 0.0063 0.1607 ± 0.0056 0.1582 ± 0.0078

L. Epididymis 0.4933 ± 0.0157 0.4944 ± 0.0084 0.4973 ± 0.0140 0.4828 ± 0.0149

L. Testis 1.5400 ± 0.0487 1.5126 ± 0.0259 1.5180 ± 0.0316 1.4909 ± 0.0441

Spermatid measurements

Spermatid heads (107

/g testis) 9.15 ± 0.42 9.51 ± 0.38 9.12 ± 0.54 9.36 ± 0.35

Spermatid heads (107

/testis) 13.98 ± 0.56 14.35 ± 0.49 13.84 ± 0.83 13.90 ± 0.49

Spermatid count

 (mean/10-4 mL suspension) 69.90 ± 2.78 71.75 ± 2.45 69.18 ± 4.16 69.48 ± 2.46

Epididymal spermatozoal measurements

Motility (%) 84.66 ± 0.43b 83.63 ± 0.47 83.32 ± 0.47 83.27 ± 0.40

Concentration

 (106

/g cauda epididymal tissue) 427 ± 19 454 ± 21 460 ± 17 409 ± 36

* Significantly different (P#0.05) from the vehicle control group by Dunnettís test a Data are presented as mean ± standard error. Differences from the vehicle control group are not significant by Dunnettís test (tissue 

weights) or Dunnís test (spermatid and epididymal spermatozoal measurements). b n=8

TABLE E2

Estrous Cycle Characterization for Female Rats in the 13-Week Gavage Study

of Benzyltrimethylammonium Chloridea

Vehicle Control 25 mg/kg 50 mg/kg 100 mg/kg

n 10 9 10 8

Necropsy body wt (g) 190 ± 3 193 ± 3 192 ± 4 187 ± 4

Estrous cycle length (days) 4.75 ± 0.13 4.44 ± 0.15b 4.80 ± 0.17 4.94 ± 0.26

Estrous stagesc

 (% of cycle)

Diestrus 42.5 36.1 38.3 37.5

Proestrus 14.2 20.4 17.5 18.8

Estrus 25.0 23.1 25.8 22.9

Metestrus 18.3 20.4 18.3 20.8

a Necropsy body weight and estrous cycle length data are presented as mean ± standard error. Differences from the vehicle control group 

are not significant by Dunnettís test (necropsy body weight) or Dunnís test (estrous cycle length). b Estrous cycle was longer than 12 days or unclear in one of nine animals. c Evidence shows that females administered 25 mg/kg differ significantly (Wilkís Criterion, P#0.05) from the vehicle control females in 

the relative length of time spent in the estrous stages. Dosed females spent more time in proestrus and less time in diestrus than the

vehicle control females.
Benzyltrimethylammonium Chloride, NTP TOX 57 E-3

TABLE E3

Summary of Reproductive Tissue Evaluations for Male Mice in the 13-Week Gavage Study

of Benzyltrimethylammonium Chloridea

Vehicle Control 25 mg/kg 50 mg/kg 100 mg/kg

n 10 10 9 9

Weights (g)

Necropsy body wt 34.9 ± 0.7 34.5 ± 0.6 35.5 ± 0.7 33.9 ± 0.9

L. Cauda epididymis 0.0175 ± 0.0008 0.0167 ± 0.0010 0.0173 ± 0.0010 0.0174 ± 0.0009

L. Epididymis 0.0503 ± 0.0016 0.0470 ± 0.0016 0.0514 ± 0.0020 0.0486 ± 0.0015

L. Testis 0.1106 ± 0.0044 0.1150 ± 0.0033 0.1180 ± 0.0052 0.1184 ± 0.0034

Spermatid measurements

Spermatid heads (107

/g testis) 15.90 ± 0.34 14.83 ± 0.44 14.25 ± 0.35* 15.10 ± 0.49

Spermatid heads (107

/testis) 1.76 ± 0.09 1.70 ± 0.05 1.69 ± 0.09 1.78 ± 0.07

Spermatid count

 (mean/10-4 mL suspension) 55.10 ± 2.76 53.05 ± 1.47 52.64 ± 2.75 55.78 ± 2.13

Epididymal spermatozoal measurements

Motility (%) 88.75 ± 0.31 88.42 ± 0.52b 88.92 ± 0.54 87.82 ± 0.40

Concentration

 (106

/g cauda epididymal tissue) 914 ± 55 925 ± 71 796 ± 117 856 ± 57

* Significantly different (P#0.05) from the vehicle control group by Dunnettís test a Data are presented as mean ± standard error. Differences from the vehicle control group are not significant by Dunnettís test (tissue 

weights) or Dunnís test (spermatid heads per testis, spermatid count, and epididymal spermatozoal measurements). b n=9

TABLE E4

Estrous Cycle Characterization for Female Mice in the 13-Week Gavage Study

of Benzyltrimethylammonium Chloridea

Vehicle Control 25 mg/kg 50 mg/kg 100 mg/kg

n 10 10 10 9

Necropsy body wt (g) 29.1 ± 1.0 28.7 ± 0.9 29.2 ± 1.3 28.2 ± 0.9

Estrous cycle length (days) 4.00 ± 0.00b 4.30 ± 0.13 4.61 ± 0.44c 4.17 ± 0.12

Estrous stages (% of cycle)

Diestrus 40.0 32.5 36.7 27.8

Proestrus 16.7 17.5 15.8 17.6

Estrus 22.5 29.2 26.7 30.6

Metestrus 20.8 20.8 20.8 24.1

a Necropsy body weight and estrous cycle length data are presented as mean ± standard error. Differences from the vehicle control group 

are not significant by Dunnettís test (necropsy body weight) or Dunnís test (estrous cycle length). By multivariate analysis of variance, 

dosed females do not differ significantly from the vehicle control females in relative length of time spent in the estrous stages. b Estrous cycle was longer than 12 days or unclear in 3 of 10 animals. c Estrous cycle was longer than 12 days or unclear in 1 of 10 animals.
E-4 Benzyltrimethylammonium Chloride, NTP TOX 57
F-1

APPENDIX F

GENETIC TOXICOLOGY

TABLE F1 Mutagenicity of Benzyltrimethylammonium Chloride in Salmonella typhimurium . . . . F-2

TABLE F2 Frequency of Micronuclei in Peripheral Blood Erythrocytes of Mice

Following Treatment with Benzyltrimethylammonium Chloride by Gavage

for 13 Weeks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F-3
F-2 Benzyltrimethylammonium Chloride, NTP TOX 57

TABLE F1

Mutagenicity of Benzyltrimethylammonium Chloride in Salmonella typhimuriuma

Revertants/plateb

Strain Dose

(μg/plate)

!S9 + hamster S9 +rat S9

Trial 1 Trial 2 10% 30% 10% 30%

TA100 0 122 ± 8.2 122 ± 9.2 116 ± 7.0 119 ± 11.0 84 ± 6.6 147 ± 9.6

100 119 ± 12.7 128 ± 3.7 102 ± 1.2 130 ± 4.9 105 ± 7.1 128 ± 7.1

333 115 ± 6.9 118 ± 14.2 110 ± 3.5 115 ± 9.0 89 ± 6.9 128 ± 3.4

1,000 116 ± 11.7 116 ± 8.2 107 ± 8.1 113 ± 5.7 104 ± 8.7 136 ± 2.5

3,333 121 ± 9.0c 122 ± 2.4 99 ± 3.0 135 ± 2.9c 102 ± 8.7 138 ± 3.8

10,000 65 ± 3.7c 104 ± 4.4c 103 ± 13.5c 109 ± 6.4c 91 ± 7.8 113 ± 7.2c

Trial summary Negative Negative Negative Negative Negative Negative

Positive controld 1,389 ± 22.6 1,485 ± 4.8 1,125 ± 21.5 2,231 ± 35.7 1,883 ± 33.0 702 ± 16.7

TA1535 0 31 ± 3.0 29 ± 0.9 9 ± 0.9 14 ± 0.9 9 ± 1.7 12 ± 2.3

100 29 ± 0.7 30 ± 1.0 11 ± 3.8 8 ± 1.0 10 ± 1.9 13 ± 0.0

333 25 ± 0.9 26 ± 4.4 13 ± 2.3 12 ± 1.5 10 ± 1.5 15 ± 2.0

1,000 28 ± 1.5 37 ± 2.2 9 ± 1.2 10 ± 1.7 11 ± 0.7 11 ± 0.7

3,333 28 ± 3.0c 27 ± 2.7 9 ± 0.9 12 ± 3.5 9 ± 3.8 13 ± 1.2

10,000 22 ± 4.8c 21 ± 1.5c 9 ± 1.5c 12 ± 0.6 11 ± 1.5c 11 ± 2.3

Trial summary Negative Negative Negative Negative Negative Negative

Positive control 1,051 ± 8.7 1,095 ± 18.9 127 ± 9.0 323 ± 17.8 117 ± 5.0 130 ± 13.9

TA97 0 104 ± 8.4 121 ± 3.5 140 ± 4.1 170 ± 8.8 113 ± 9.7 181 ± 5.5

100 113 ± 7.0 110 ± 7.4 143 ± 0.3 158 ± 8.8 126 ± 12.1 181 ± 4.2

333 99 ± 0.9 114 ± 3.6 144 ± 8.0 156 ± 1.9 124 ± 3.5 155 ± 9.2

1,000 112 ± 8.3 128 ± 5.5 139 ± 6.9 165 ± 6.1 133 ± 5.3 168 ± 11.1

3,333 95 ± 2.1c 128 ± 2.9 130 ± 7.1 152 ± 6.0c 129 ± 2.2 167 ± 6.4

10,000 92 ± 5.2c 100 ± 5.6c 131 ± 2.2c 172 ± 7.8c 110 ± 4.6c 174 ± 12.8

Trial summary Negative Negative Negative Negative Negative Negative

Positive control 1,626 ± 161.0 722 ± 9.3 511 ± 17.2 1,452 ± 23.1 763 ± 36.3 691 ± 13.3

TA98 0 19 ± 4.1 16 ± 0.6 39 ± 2.6 35 ± 2.7 32 ± 5.5 31 ± 5.8

100 14 ± 1.5 14 ± 1.7 37 ± 3.4 27 ± 4.4 31 ± 1.2 29 ± 0.9

333 16 ± 2.1 19 ± 4.1 32 ± 3.2 31 ± 2.8 30 ± 1.3 34 ± 2.6

1,000 17 ± 2.0 17 ± 2.4 36 ± 2.1 34 ± 1.2 35 ± 1.7 30 ± 5.8

3,333 19 ± 1.2c 16 ± 1.5 33 ± 5.5 30 ± 3.5 38 ± 2.2 35 ± 2.2

10,000 14 ± 1.0c 19 ± 3.5c 34 ± 1.2 28 ± 1.8 31 ± 3.8 36 ± 1.7c

Trial summary Negative Negative Negative Negative Negative Negative

Positive control 1,246 ± 69.5 1,848 ± 57.8 1,205 ± 26.9 2,210 ± 28.6 1,401 ± 30.6 659 ± 32.9

a Study was performed at Microbiological Associates, Inc. The detailed protocol and these data are presented in Zeiger et al. (1988). 

0 μg/plate was the solvent control. b Revertants are presented as mean ± standard error from three plates. c Slight toxicity d The positive controls in the absence of metabolic activation were sodium azide (TA100 and TA1535), 9-aminoacridine (TA97), and

4-nitro-o-phenylenediamine (TA98). The positive control for metabolic activation with all strains was 2-aminoanthracene.
Benzyltrimethylammonium Chloride, NTP TOX 57 F-3

TABLE F2

Frequency of Micronuclei in Peripheral Blood Erythrocytes of Mice Following Treatment

with Benzyltrimethylammonium Chloride by Gavage for 13 Weeksa

Compound Dose

(mg/kg)

Number of Mice

with Erythrocytes

Scored

Pairwise

P Value Micronucleated NCEs/1,000 NCEsb c

Male

Waterd 10 3.7 ± 0.6

Benzyltrimethylammonium chloride 12.5 10 2.5 ± 0.5 0.937

25 10 2.8 ± 0.6 0.868

50 10 5.2 ± 0.9 0.056

100 9 6.6 ± 1.1 0.003

P#0.001e

Female

Water 10 2.0 ± 0.3

Benzyltrimethylammonium chloride 12.5 10 2.5 ± 0.6 0.228

25 10 3.0 ± 0.3 0.078

50 10 3.9 ± 0.3 0.007

100 9 6.4 ± 0.6 0.000

P#0.001

a Study was performed at Integrated Laboratory Systems. The detailed protocol is presented in MacGregor et al. (1990). 

NCE=normochromatic erythrocyte b Mean ± standard error c Pairwise comparison to solvent control; significant at P#0.006 (ILS, 1990) d Solvent control e Significance of micronucleated NCEs/1,000 NCEs tested by the one-tailed trend test, significant at P#0.025 (ILS, 1990)
F-4 Benzyltrimethylammonium Chloride, NTP TOX 57
NTP TECHNICAL REPORTS ON TOXICITY STUDIES

PRINTED AS OF FEBRUARY 2000

TOX No. Chemical TOX No. Chemical

1 Hexachloro-1,3-butadiene 28 Tetrachlorophthalic Anhydride

2 n-Hexane 29 Cupric Sulfate

3 Acetone 30 Dibutyl Phthalate

4 1,2-Dichloroethane 31 Isoprene

5 Cobalt Sulfate Heptahydrate 32 Methylene Bis(thiocyanate)

6 Pentachlorobenzene 33 2-Chloronitrobenzene and 4-Chloronitrobenzene

7 1,2,4,5-Tetrachlorobenzene 34 1-Nitropyrene

8 D & C Yellow No. 11 35 Chemical Mixture of 25 Groundwater Contaminants

9 o-Cresol, m-Cresol, and p-Cresol 36 Pesticide/Fertilizer Mixtures

10 Ethylbenzene 37 Sodium Cyanide

11 Antimony Potassium Tartrate 38 Sodium Selenate and Sodium Selenite

12 Castor Oil 39 Cadmium Oxide

13 Trinitrofluorenone 40 $-Bromo-$-nitrostyrene

14 p-Chloro-",","-trifluorotoluene 42 1,3-Diphenylguanidine

15 t-Butyl Perbenzoate 43 o-, m-, and p-Chloroaniline

16 Glyphosate 44 o-Nitrotoluene and o-Toluidine Hydrochloride

17 Black Newsprint Ink 45 Halogenated Ethanes

18 Methyl Ethyl Ketone Peroxide 50 Cyclohexanone Oxime

19 Formic Acid 51 Methyl Ethyl Ketoxime

20 Diethanolamine 52 Urethane

21 2-Hydroxy-4-methoxybenzophenone 53 t-Butyl Alcohol

22 N, N-Dimethylformamide 54 1,4-Butanediol

23 o-Nitrotoluene, m-Nitrotoluene, and p-Nitrotoluene 58 60-Hz Magnetic Fields

24 1,6-Hexanediamine 59 Chloral Hydrate

25 Glutaraldehyde 65 3,3N,4,4N-Tetrachloroazobenzene

26 Ethylene Glycol Ethers 66 3,3N,4,4N-Tetrachloroazoxybenzene

27 Riddelliine