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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 October 1993 -12 January 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study appeared to be conducted according to test guidelines and in accordance with GLP

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2000

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
not specified
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
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 manufacturer's stability information, the bulk chemical was stored at room temperature in sealed containers flushed with nitrogen to expel moisture.

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

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
Male and female F344/N rats were obtained from Taconic Farms (Germantown, NY). Upon receipt, the rats 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 were randomly selected for parasite evaluation and gross observation for evidence of disease. Blood was collected from five male and five female control rats 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). All results were negative.

Feed and water were available ad libitum. Rats were housed five per cage.

References:
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.

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.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
Core study groups of 10 male and 10 female rats 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.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
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 amber glass bottles (13-week studies) at room temperature.

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 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 all animal room samples were within 10% of the target concentrations.
Duration of treatment / exposure:
5 days per week for 13 weeks.
Frequency of treatment:
Daily, except weekends
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 12.5, 25, 50, or 100 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
10 males and 10 females/dose level
Control animals:
yes, concurrent vehicle
Details on study design:
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.
Positive control:
No data.

Examinations

Observations and examinations performed and frequency:
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. 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.

Sacrifice and pathology:
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 hematology and clinical chemistry analyses were the same as those used in the 16-day studies. 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.

A necropsy was performed on all core study rats. 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. 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 Zymbals gland (rats). The lung of all 50 mg/kg rats was also examined.

Upon completion of the laboratory pathologist's 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.
Other examinations:
At the end of the 13-week studies, samples were collected for sperm motility and vaginal cytology evaluations on core study rats receiving 0, 25, 50, or 100 mg/kg. 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.Methods used were those described in the NTP's 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) 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 65 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.
Reference:
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.
Statistics:
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). Jonckheeres test (Jonckheere, 1954) was used to assess the significance of the dose-related trends and to determine whether a trend-sensitive test (Williams' or Shirley's test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose-related trend (Dunnett's or Dunn's 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).

Calculation and Analysis of Lesion Incidences
The incidences of lesions 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 animals, was used to determine

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Two 100 mg/kg female rats died before the end of the study. Nasal and eye discharge in some 25 - 100 mg/kg males and 50 - 100 mg/kg females and tremors in 100 mg/kg animals were observed.
Mortality:
mortality observed, treatment-related
Description (incidence):
Two 100 mg/kg female rats died before the end of the study. Nasal and eye discharge in some 25 - 100 mg/kg males and 50 - 100 mg/kg females and tremors in 100 mg/kg animals were observed.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Decreased body weight gain in 100 mg/kg males
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not specified
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Altered gait and tremors observed in some rats from the 100 mg/kg group. Clinical evaluation demonstrated chromodacryorrhea and increased salivation in rats in the 100 mg/kg group.
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not specified
Details on results:
One 25 mg/kg and two 100 mg/kg female rats died before the end of the study; 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. Chemical related 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).

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. 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.

Significant differences were observed in the hematology and clinical chemistry variables. 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 administration had no effect on the absolute or relative organ weights of males or females. No chemical-related gross or microscopic lesions were observed. There were no differences in reproductive tissue parameters in males. 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.

Effect levels

open allclose all
Dose descriptor:
LOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Mortality and cholinergic effects observed.
Dose descriptor:
NOAEL
Effect level:
25 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

No additional information available.

Applicant's summary and conclusion

Conclusions:
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.
Executive summary:

The subchronic toxicity of benzyltrimethylammonium chloride was examined in F344 rats. In the 13 -week studies, groups of 10 male and 10 female rats 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. Final mean body weights of dosed animals were within 8% (rats) of the control group body weights. The deaths of two female rats 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.

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.