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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Benzyltrimethylammonium chloride
- Molecular formula (if other than submission substance): C10H16ClN
- Molecular weight (if other than submission substance): 185.7
- Analytical purity: The purity of the test material was determined to be 99.9% (anhydrous basis) by high performance liquid chromatography with identification by nuclear magnetic resonance and liquid chromatography mass spectrometry
- Lot/batch No.:Lot# ZMG-184366
- Stability under test conditions: A test material purity, method validation, solubility and stability study with BTMAC in ultrapure water is being conducted concurrent with this study. The concentration ranges tested in the stability study span the concentrations planned for this study, and the dose solutions will be used within the established stability limits. The method used for analyzing the test material in the vehicle is HPLC with UV detection.

Test animals

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

Administration / exposure

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
Doses / concentrationsopen allclose all
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.

Examinations

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.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

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.

Effect levels (maternal animals)

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

Results (fetuses)

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.

Effect levels (fetuses)

Dose descriptor:
NOEL
Effect level:
20 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: embryotoxicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.