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Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
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.
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
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.
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.
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.
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
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: 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
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.
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
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
Rats were not bred and therefore no offspring were part of this study.
Reproductive effects observed:
not specified

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.

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.
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Studies are guideline, conducted by the US National Toxicology Program
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In two 90-day studies in rodents, no evidence of histologic changes in reproductive organs was seen in either sex. In addition, no consistent, dose-related effects on estrous cyclicity were noted in either species.


Short description of key information:
No evidence of reproductive effects in 90-day studies in both rats and mice - estrous cyclicity also assessed.

Justification for selection of Effect on fertility via oral route:
Both studies were negative for effects. Rat study chosen as preferred species.

Effects on developmental toxicity

Description of key information
In this prenatal developmental toxicity study, the no-observed-effect level (NOEL) for maternal toxicity was 20 mg/kg bw/day, based on a decrease in maternal body weight gain throughout the treatment period, decreased feed consumption, decrease in terminal body weight and absolute spleen weight. 
The embryo/fetal NOEL was 20 mg/kg bw/day, based on decreases in mean fetal body weights (male, female and combined), and consequently lower mean gravid uterine weights.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Follows GLP and OECD guidelines
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (Raleigh, North Carolina)
- Age at study initiation: Sexually mature adults
- Weight at study initiation: 200-250 g
- Housing: After assignment, animals will be housed one per cage in stainless steel cages. Cages will have solid floors with corncob bedding. Cages will contain a feed crock and a pressure activated lixit valve-type watering system.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: Animals were acclimated to the laboratory for at least four days prior to the start of dosing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C with a range of 20°C-26°C
- Humidity (%): 50% with a range of 30-70%
- Air changes (per hr): 10-15 times/hour (average)
- Photoperiod (hrs dark / hrs light): 12-hour light/dark (on at 6:00 a.m. and off at 6:00 p.m.)

IN-LIFE DATES: From: April 26,2015 To: May 27,2015
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
BTMAC solutions were prepared in ultrapure water at concentrations of 0, 8, 12, and 16 mg/ml and administered at a dose volume of 5 ml/kg body weight in order to achieve the targeted dose levels. Dose solutions were not corrected for purity. Dose volumes were adjusted daily based on individual body weights. The control rats were dosed with ultrapure water at 5 ml/kg body weight. Dose solutions were prepared periodically throughout the study.

VEHICLE
Ultrapure water (The Dow Chemical Company, Midland, Michigan).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Solubility:
BTMAC was determined to be soluble in ultrapure water at target concentrations of up to 500 mg/mL.

Concentration Verification and Homogeneity
Dose confirmation analyses of all dose levels, plus control, were determined pre-exposure. The low- and high-dose solutions from the first mix were analyzed prior to the start of dosing to verify homogeneous distribution of the test material in vehicle. The method used for analyzing the test material in the vehicle was high performance liquid chromatography (HPLC) with ultraviolet (UV) detection

Stability:
A test material purity, method validation, solubility and vehicle stability study with BTMAC in ultrapure water was conducted concurrently with this study (McFadden, 2015). The concentration ranges tested in the stability study spanned the concentrations and durations planned for this study, and the dose solutions were used within the established 16-day vehicle stability limit. The method used for analyzing the test material in the vehicle was high performance liquid chromatography (HPLC) with ultraviolet (UV) detection.
Details on mating procedure:
Sexually mature, adult virgin females were naturally mated with males of the same strain (one male:one female) at the suppliers facility. Females were checked for in situ copulation plugs the following morning, and those found with such a plug were removed from the males’ cages. The day on which a vaginal plug was detected was considered GD 0. GD 0 body weights were provided by the supplier and maintained in the study record. Rats arrived in our laboratory on GD 1 or 2.
Duration of treatment / exposure:
Groups of 24 time-mated female Crl:CD(SD) rats were administered BTMAC in ultrapure water by gavage at targeted dose levels of 0, 5, 20, or 80 mg/kg/day on gestation day (GD) 6 through 20.
Frequency of treatment:
once daily, seven days/week from GD 6-20.
Duration of test:
GD 6-20
Remarks:
Doses / Concentrations:
0 mg/kg/day
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
5 mg/kg/day
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
20 mg/kg/day
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
80 mg/kg/day
Basis:
nominal conc.
No. of animals per sex per dose:
24 female per
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels for this study (Text Table 1) were selected on the basis of the developmental toxicity probe study discussed previously (see Previous Toxicity section). Based on the results of the probe study, the high dose of 80 mg/kg/day was selected and was expected to induce overt signs of maternal toxicity including a decrease in maternal body weight gain and feed consumption. The lower dose levels were selected to provide dose response data for any toxicity that may be observed among the high-dose group rats.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes


POST-MORTEM EXAMINATIONS: Yes


OTHER:
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Fetal examinations:
- External examinations: Yes:
- Soft tissue examinations: Yes:
- Skeletal examinations: Yes:
- Head examinations: Yes:
Statistics:
Maternal body weights, maternal body weight gains, organ weights (absolute and relative), fetal body weights and feed consumption were evaluated by Bartlett’s test (alpha = 0.01; Winer, 1971) for equality of variances. Based on the outcome of Bartlett's test, a parametric (Steel and Torrie, 1960) or nonparametric (Hollander and Wolfe, 1973) analysis of variance (ANOVA) was performed. If the ANOVA was significant at alpha = 0.05, analysis by Dunnett's test (alpha = 0.05; Winer, 1971) or the Wilcoxon Rank-Sum test (alpha = 0.05; Hollander and Wolfe, 1973) with Bonferroni's correction (Miller, 1966) was performed, respectively. Feed consumption values were excluded from analysis if the feed was spilled or scratched.
Frequency of pre- and post-implantation loss (calculations shown below), and fetal alterations (if any) were analyzed using a censored Wilcoxon test (Haseman and Hoel, 1974) with Bonferroni’s correction applied when the incidence was greater than 5%. The number of corpora lutea, implantations, and litter size were evaluated using a nonparametric ANOVA (alpha = 0.05) followed by the Wilcoxon Rank-Sum test (alpha = 0.05) with Bonferroni's correction. Pregnancy rates were analyzed using the Fisher exact probability test (alpha = 0.05; Siegel, 1956) with Bonferroni’s correction. Fetal sex ratios were analyzed using a binomial distribution test. Non-pregnant females, females with resorptions only, or females found to be pregnant after staining of their uteri were excluded from the appropriate analyses. Statistical outliers (alpha = 0.02) were identified by the sequential method of Grubbs (1969) and were only excluded from analysis for documented, scientifically sound reasons. Both Dunnett’s test and Bonferroni’s correction correct for multiple comparisons to the control group to keep the experiment-wise alpha at 0.05. Both were reported at the experiment-wise alpha level.
Historical control data:
Developmental Toxicity Probe Study
In a rat developmental toxicity probe study (Zablotny et al., 2015), groups of five time-mated female Cr1:CD(SD) rats were administered 0, 40, 60, or
80 mg/kg/day BTMAC in ultrapure water by oral gavage at a dose volume of 5 ml/kg on gestation day (GD) 6 through 20. In-life parameters evaluated for all groups included clinical observations, body weight, body weight gain, and feed consumption. On GD 21 all surviving dams were euthanized and examined for gross pathologic alterations. Liver, kidney and spleen weights were recorded, along with the number of corpora lutea, implantations, resorptions, and live/dead fetuses.
Oral gavage administration of BTMAC to Crl:CD(SD) rats resulted in maternal toxicity in the 80 mg/kg/day group as evidenced by a treatment-related 10% decrease in maternal body weight gain throughout the treatment period (GD 6-21) and a 23% decrease during GD 18-21. These body weight gain effects correlated with lower feed consumption. There were no treatment-related effects on body weights, body weight gain, or feed consumption in the 40 or 60 mg/kg/day groups.
There was a treatment-related decrease in absolute and relative spleen weight in the
80 mg/kg/day group. There were no treatment-related effects on spleen weights in the 40 or 60 mg/kg/day dose groups, or on liver or kidney weights at any dose levels tested. There were no treatment-related gross pathologic changes in females at any dose levels tested.
There were no effects on any measured parameter of developmental toxicity including embryo/fetal lethality in animals at any dose level tested.
Based upon the treatment-related decrease in body weight gain observed at 80 mg/kg/day in this study, BTMAC oral gavage dose levels ≤ 80 mg/kg/day may be considered for a full prenatal developmental toxicity study in Crl:CD(SD) rats.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Mortality
One dam (2226) given 80 mg/kg/day was found dead on GD 10. The cause of death was not determined (See Anatomic Pathology; Histopathology Unscheduled death). All other animals assigned to the study survived until scheduled termination.
In-Life Observations
Examinations performed pre-exposure revealed that all animals were in good health for study purposes. During the treatment period, clear and/or red perioral soiling were observed in nine and five dams in the 80 mg/kg/day dose group, respectively (Text Table 3). In addition, decreased activity was observed following dosing in eight dams given 80 mg/kg/day. In general, these observations were most prevalent towards the end of gestation (GD 17-20). All animals that had decreased activity or clear or red perioral soiling on GD 20 had no remarkable observations on GD 21 prior to necropsy. The observations noted were consistent with other previously studies conducted with BTMAC and deemed treatment related (National Toxicology Program, 2000). All other observations were considered incidental and bore no relationship to treatment.
Body Weights/Body Weight Gains
When compared to controls, animals administered 80 mg/kg/day BTMAC had a treatment-related decrease in maternal body weight gain during the GD 6-9, 9-12, 12-15, 18-21, and 0-21 intervals, along with a 20.1% reduction in body weight gain over the GD 6-21 period (Text Table 4). Additionally, the largest decrease in body weight gain (37%) occurred during the GD 18-21 interval. The body weight gain decrements in the 80 mg/kg/day dose group correlated with decreases in feed consumption during the same intervals (Text Table 6). Animals in the 80 mg/kg/day group also had statistically identified, and treatment-related GD 21 mean body weight and GD 21 corrected mean body weight (GD 21 body weight – gravid uterine weight) decreases of 8.3 and 6.8%, respectively (Text Table 5). There were no treatment-related decreases in gestation body weights or body weight gains in the 5 or 20 mg/kg/day dose groups.
Feed Consumption
Animals in the 80 mg/kg/day group had statistically significant, treatment-related decreases in feed consumption during the GD 6-9, 12-15, 15-18 and 18-21 intervals that ranged from 9.7-18.3% lower than controls with the largest decrement occurring during the GD 18-21 interval (Text Table 6). There were no treatment-related effects on feed consumption in animals in the 5 and 20 mg/kg/day groups when compared to control.
Anatomic Pathology
Females administered 80 mg/kg/day had statistically significant, treatment-related decreases in terminal body weight (8.3%) and absolute spleen weights (13.6%) relative to control (Text Table 7). There were no treatment-related effects on relative spleen weights at 80 mg/kg/day or absolute and relative spleen weights in the 5 or 20 mg/kg/day dose groups. There were no treatment-related effects on absolute liver or kidney weights at any dose level tested. Females given 80 mg/kg/day had higher mean relative liver and kidney weights that were statistically significant, and interpreted to be reflective of the lower body weights of females at this dose level. There were no treatment-related effects on relative liver or kidney weights in the 5 or 20 mg/kg/day dose groups when compared to control.
Gross Pathology
A summary of gross pathologic observations are presented in Table 8 for animals that died spontaneously or absorbed their litters prior to scheduled necropsy. Individual data are reported in Appendix Table 6.
There were no treatment-related gross pathologic observations in any dose level.
Histopathology – Unscheduled Death
Animal 2226 from the 80 mg/kg/day group was found dead on GD 10. At necropsy this animal had liver and kidney congestion and mottled lungs (Appendix Table 6). Histopathological alterations in the lungs of this animal included slight congestion. There was no evidence of gavage error (Appendix Table 6). The cause of death for this animal was not determined and therefore, the relationship to treatment remains equivocal.
Reproductive Parameters
There were no treatment related effects on pregnancy rates, resorption rates, litter size, numbers of corpora lutea or implantations, percent preimplantation loss, percent postimplantation loss, or fetal sex ratios at any dose level.
The mean male, female, and combined fetal body weights in the 80 mg/kg/day group were decreased (3.7 -4.4%) relative to control with the mean male fetal body weights statistically identified (Text Table 8). All of these values are slightly below the laboratory historical control range (Text Table 9). These treatment-related decreases in fetal body weights are likely secondary to the treatment-related decreases in maternal feed consumption and body weight gain in the 80 mg/kg/day dose group. Although not statistically identified, the gravid uterine weights in animals administered 80 mg/kg/day were decreased 12.7% relative to control (Text Table 8). This treatment-related effect is outside the laboratory historical control range (Text Table 9). There were no treatment-related effects on fetal body weights or gravid uterine weights in the 5 or 20 mg/kg/day dose groups.

Dose descriptor:
NOEL
Effect level:
20 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Compared to controls, there were no treatment-related differences in the incidence of any fetal alteration in any of the treated groups. The small number of alterations observed in fetuses from dams administered BTMAC either occurred at low frequencies and/or were not dose related.
External Examination
There were no treatment-related external alternations in any dose group. Incidental findings bearing no relationship to treatment included the malformation gastroschisis umbilicus in one fetus (dam #2216) in the 80 mg/kg/day dose group (Text Table 10). Given that this observation occurred in a single animal, this observation was considered spurious and unrelated to treatment
Craniofacial Examination
There were no treatment-related craniofacial alternations in any dose group. Incidental findings bearing no relationship to treatment included the malformation dilated cerebral ventricles in one fetus (dam #2216) in the 80 mg/kg/day dose group (Text Table 11). Given that this observation occurred in a single animal, this observation was considered spurious and unrelated to treatment.
Visceral Examination
There were no treatment-related visceral alternations in any dose group. An Incidental findings bearing no relationship to treatment included the malformation misshapen liver (Text Table 12) and the variations hemorrhage liver, supernumerary hepatic lobule, bifurcated renal vein, and no meconium intestine. Given that these observations occurred in the control group, at low frequencies, and/or lacked a dose response, these observations were considered spurious and unrelated to treatment.
Skeletal Examination
There were no treatment-related skeletal alternations in any dose group. Incidental findings bearing no relationship to treatment included the malformations fused ribs, and missing lumbar centra and vertebra (Text Table 13) and the variations DO interparietal, DO thoracic centra, DO sternebrae, irregular pattern of ossification sternebrae, calloused ribs, class I and II wavy ribs, and extra 1st lumbar rib. Given that these observations occurred in the control group, at low frequencies, and/or lacked a dose response, these observations were considered spurious and unrelated to treatment. Although the incidence of DO sternebrae appeared elevated to control in the 80 mg/kg/day dose group (Text Table 14), the incidence was deemed spurious and unrelated to treatment since the incidence was not statistically identified and was within the laboratory historical control range.
Dose descriptor:
NOEL
Effect level:
20 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: embryotoxicity
Abnormalities:
not specified
Developmental effects observed:
not specified

Selected Reproductive Parameters

 Dose Level (mg/kg/day)  0  5  20  80
 Fetal Weight- Males (g)  5.93  6.13  6.01  5.67* (-4.4%)
 Fetal Weight- Females (g)  5.67  5.82  5.68  5.46 (-3.7%)
 Fetal Weight -Sexes combined (g)  5.80  5.98  5.83  5.57 (-4.0%)
 Gavid Uterine Weight (g)  105.3  99.7  99.9  91.9 (-12.7%)

*Statistically different from control mean by Dunnett’s test, alpha = 0.05.

Percentages are the percent difference from controls

 Boldtype indicates the effects judged to be treatment related.

Incidences of External Malformations:

 Dose (mg/kg/day)  0  5  20  80
  Gastroschsis Umbilicus  F   0/305  0/291  0/298  1/264a
   L  0/23  0/24  0/24  1/22

F = fetuses; L = litters

aMalformations denoted with the same superscript were noted in a single fetus and were observed in the same fetus with malformations noted.

Incidences of Visceral Malformations

Dose (mg/kg/day)     0  5  20  80
 Misshapen Liver  F  0/153  0/145  0/151  1/134a
   L  0/23  0/24  0/24  1/22

F = fetuses; L = litters

aMalformations denoted with the same superscript were noted in a single fetus and were observed in the same fetus with malformations noted

Incidences of Skeletal Malformations

 Dose (mg/kg/day)    0  5  20  80
 Fused Ribs  F  1/152  0/146  0/146  0/130
   L  1/23  0/24  0/24  0/22
 Missing Lumbar Centra  F  1/152a  0/146  0/146  0/130
   L  1/23  0/24  0/24  0/22
 Missing Lumber  F  1/152a  0/146  0/146  0/130
   L  1/23  0/24  0/24  0/22

F = fetuses; L = litters

aMalformations denoted with the same superscript were noted in a single fetus.

Incidences of Skeletal Variation (DO Sternebrae)

 Dose (mg/kg/day)    0  5  20  80
 DO Sternebrae  F  0/152  1/146  1/146  3/130
   L  0/23  1/24  1/24  2/22

F = fetuses; L = litters

Laboratory Historical Control values range from 0 -3 for Incidence of DO Sternebrae in fetuses and from 0 -2 for Incidence of DO Sternebrae in litters.

Conclusions:
Under the conditions of this study, the no-observed-effect level (NOEL) for maternal toxicity was 20 mg/kg/day, and the embryo/fetal NOEL was 20 mg/kg/day.
Executive summary:

The purpose of this study was to evaluate the maternal and developmental toxicity of benzyltrimethylammonium chloride (hereafter referred to as BTMAC) in Crl:CD(SD) rats following repeated gavage administration. Groups of 24 time-mated female Crl:CD(SD) rats were administered BTMAC in ultrapure water by gavage at targeted dose levels of 0, 5, 20, or 80 mg/kg/day on gestation day (GD) 6 through 20. In-life maternal study parameters included clinical observations, body weight, body weight gain and feed consumption. On GD 21, all surviving rats were euthanized and examined for gross pathologic alterations. Liver, kidneys, spleen, and gravid uterine weights were recorded, along with the number of corpora lutea, uterine implantations, resorptions, and live/dead fetuses. All fetuses were weighed, sexed and examined for external alterations. Approximately one half of the fetuses were examined for visceral alterations while skeletal examinations were conducted on the remaining fetuses.

Maternal toxicity was limited to dams given 80 mg/kg/day and consisted of a treatment-related increase in the incidences of clear or red perioral soiling and decreased activity following dosing accompanied by a 20.1% decrease in maternal body weight gain throughout the treatment period (GD 6-21) and a 37% decrease during GD 18-21. These body weight gain effects correlated with decreased feed consumption. At necropsy,there was a treatment-related decrease in terminal body weight and absolute spleen weight in the 80 mg/kg/day dose group. Relative liver and kidney weights in the 80 mg/kg/day dose group were increased and deemed secondary to the lower terminal body weight. 

The maternal effects at 80 mg/kg/day resulted in decreases in mean fetal body weights (male, female and combined), and consequently lower mean gravid uterine weights. There were no other effects on fetal development at this dose level. There was no treatment-related maternal or developmental toxicity in the 5 or 20 mg/kg/day dose groups.

Therefore, under the conditions of this study, the no-observed-effect level (NOEL) for maternal toxicity was 20 mg/kg/day, and the embryo/fetal NOEL was 20 mg/kg/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
20 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Dose range finding study

In a rat developmental toxicity probe study (Zablotny et al., 2015), groups of five time-mated female Cr1:CD(SD) rats were administered 0, 40, 60, or 80 mg/kg bw/day BTMAC in ultrapure water by oral gavage at a dose volume of 5 ml/kg on gestation day (GD) 6 through 20. In-life parameters evaluated for all groups included clinical observations, body weight, body weight gain, and feed consumption. On GD 21 all surviving dams were euthanized and examined for gross pathologic alterations. Liver, kidney and spleen weights were recorded, along with the number of corpora lutea, implantations, resorptions, and live/dead fetuses.

Oral gavage administration of BTMAC to Crl:CD(SD) rats resulted in maternal toxicity in the 80 mg/kg bw/day group as evidenced by a treatment-related 10% decrease in maternal body weight gain throughout the treatment period (GD 6-21) and a 23% decrease during GD 18-21. These body weight gain effects correlated with lower feed consumption. There were no treatment-related effects on body weights, body weight gain, or feed consumption in the 40 or 60 mg/kg bw/day groups.

There was a treatment-related decrease in absolute and relative spleen weight in the 80 mg/kg bw/day group. There were no treatment-related effects on spleen weights in the 40 or 60 mg/kg bw/day dose groups, or on liver or kidney weights at any dose levels tested. There were no treatment-related gross pathologic changes in females at any dose levels tested.

There were no effects on any measured parameter of developmental toxicity including embryo/fetal lethality in animals at any dose level tested.

Based upon the treatment-related decrease in body weight gain observed at 80 mg/kg bw/day in this study, BTMAC oral gavage dose levels ≤ 80 mg/kg bw/day were used for the full prenatal developmental toxicity study in Crl:CD(SD) rats.

 

Definitive study

In a prenatal developmental toxicity study according to OECD Guideline 414 BTMAC (99.9% a.i.) in ultrapure water was administered to groups of 24 time-mated female Crl:CD(SD) rats by gavage at targeted dose levels of 0, 5, 20, or 80 mg/kg bw/day on gestation day (GD) 6 through 20.

Sexually mature, adult virgin females were naturally mated with males of the same strain (one male:one female) at the suppliers facility. Females were checked for in situ copulation plugs the following morning, and those found with such a plug were removed from the males’ cages. The day on which a vaginal plug was detected was considered GD 0. GD 0 body weights were provided by the supplier and maintained in the study record. Rats arrived in the testing laboratory on GD 1 or 2.

Maternal toxicity was limited to dams given 80 mg/kg bw/day and consisted of a treatment-related increase in the incidences of clear or red perioral soiling and decreased activity following dosing accompanied by a 20.1% decrease in maternal body weight gain throughout the treatment period (GD 6-21) and a 37% decrease during GD 18-21. These body weight gain effects correlated with decreased feed consumption. At necropsy, there was a treatment-related decrease in terminal body weight and absolute spleen weight in the 80 mg/kg bw/day dose group. Relative liver and kidney weights in the 80 mg/kg bw/day dose group were increased and deemed secondary to the lower terminal body weight. 

The maternal effects at 80 mg/kg bw/day resulted in decreases in mean fetal body weights (male, female and combined), and consequently lower mean gravid uterine weights. There were no other effects on fetal development at this dose level. There was no treatment-related maternal or developmental toxicity in the 5 or 20 mg/kg bw/day dose groups.

Therefore, under the conditions of this study, the no-observed-effect level (NOEL) for maternal toxicity was 20 mg/kg bw/day, and the embryo/fetal NOEL was 20 mg/kg bw/day.

Justification for classification or non-classification

No evidence of reproductive effects seen to this point, therefore no classification is suggested.