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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
toxicity to reproduction
Remarks:
other: 13-week oral study
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:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
other: 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.

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 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). 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.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
See repeated dose section.
Details on mating procedure:
Not applicable.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
See repeated dose section.
Duration of treatment / exposure:
See repeated dose section.
Frequency of treatment:
See repeated dose section.
Details on study schedule:
See repeated dose section.
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
Control animals:
yes, concurrent vehicle
Details on study design:
See repeated dose section
Positive control:
No data.

Examinations

Parental animals: Observations and 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. Methods used were those described in the NTP's sperm morphology and vaginal cytology evaluations protocol (NTP, 1991).

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.
Oestrous cyclicity (parental animals):
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).
Sperm parameters (parental animals):
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 Tyrodes 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.
Litter observations:
Not applicable.
Postmortem examinations (parental animals):
See repeated dose section
Postmortem examinations (offspring):
Not applicable.
Statistics:
See repeated dose section.

Spermatid, and epididymal spermatozoal data, which have typically skewed distributions, were analyzed using the nonparametric multiple comparison methods of Shirley (1977) and Dunn (1964).

References:
Dunn, O.J. (1964). Multiple comparisons using rank sums. Technometrics 6, 241-252.

Shirley, E. (1977). A non-parametric equivalent of Williams test for contrasting increasing dose levels of a treatment. Biometrics 33, 386-389.
Reproductive indices:
Not applicable.
Offspring viability indices:
Not applicable

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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.
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):
effects observed, treatment-related
Description (incidence and severity):
Decreased body weight gain in 100 mg/kg males
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
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.
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
not examined

Details on results (P0)

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 (P0)

Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: no differences in reproductive tissue parameters in males; minimal shortening of diestrus and prolongation of proestrus occurred in 25 mg/kg bw/d females; there was no alteration in the length of the estrous cycle

Results: F1 generation

General toxicity (F1)

Clinical signs:
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined

Details on results (F1)

Rats were not bred and therefore no offspring were part of this study.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

TABLE 1 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 weight  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 Dunnetts test

a Data are presented as mean ± standard error. Differences from the vehicle control group are not significant by Dunnetts test (tissue

weights) or Dunns test (spermatid and epididymal spermatozoal measurements).

b n=8

TABLE 2 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 Dunnetts test (necropsy body weight) or Dunns 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 (Wilks 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.

Applicant's summary and conclusion

Conclusions:
In a 13 week oral gavage study in rats, 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.
Executive summary:

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.

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.