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Repeated dose toxicity: oral

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Administrative data

sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference Type:
Report date:

Materials and methods

Test guideline
equivalent or similar to guideline
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Clinical biochemistry and ophthalmological examination was not conducted
GLP compliance:
Food and Drug Administration (FDA) Good Laboratory Practice Regulations (21 CFR, Part 58)
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
Hyoscine hydrobromide trihydrate
Cas Number:
Molecular formula:
C17 H21 N O4 . Br H . 3 H2 O
Hyoscine hydrobromide trihydrate
Test material form:
solid: particulate/powder
Details on test material:
- Molecular formula (if other than submission substance): C17H22BrNOx3H2O
- Molecular weight (if other than submission substance): 438.33
- Analytical purity: 89% scopolamine hydrobromide and 11 % water- Identification test date: IRS 1985-03-28, NMR 1985-03-27
- Lot/batch No.: 14188 (obtained from Rebeco Chemicals, Inc. (New York, NY)
- Stability: Stability of the bulk chemical was monitored at the beginning of the 16-day studies and at the beginning and end of the 14-week studies using HPLC. No degradation of the bulk chemical was detected.- Storage condition of test material: To ensure stability, the bulk chemical was stored in amber glass jars at approximately 25°C under a nitrogen headspace.

Test animals

other: F344/N
Details on test animals or test system and environmental conditions:
- Source: Simonsen Laboratories, Inc. (Gilroy, CA)
- Age at study initiation: approximately 6 weeks
- Average age at Necropsy: approximately 19 to 20 weeks
- Weight at study initiation: mean body weight: male 114 to 116 +-3 g, female 95+-1 to 98+-2g
- Housing: 5 per cage; Cages: Polycarbonate (Lab Products Inc., Maywood, NJ), changed twice weekly; Bedding: Beta Chips® (Northeastern Products Co., Warrensburg, NY); Cage filters: Reemay® spun-bonded polyester (Andico, Birmingham, AL); Racks: Stainless steel (Lab Products Inc., Maywood, NJ), rotated once every 2 weeks
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 12 days
- Temperature (°C): 20 to 25°C- Humidity (%): 45 to 66%
- Air changes (per hr): minimum of 10 changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours/day fluorescent light

Administration / exposure

Route of administration:
oral: gavage
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Stability studies of the 11 and 0.2 mg/mL dose formulations were conducted by the analytical chemistry laboratory. For the 11 mg/mL formulation, samples were prepared by dissolving scopolamine hydrobromide trihydrate in deionized water and diluting to volume. Aliquots (5 mL) were mixed with internal standard solution (0.4 mg/mL acetophenone in acetonitrile) and were further diluted with 0.007 M aqueous sodium heptanesulfonic acid. The samples were then analyzed by HPLC using a Waters C18 Nova Pak column, with a mobile phase of water:acetonitrile (70:30), at a flow rate of 1.0 mL/minute, and with ultraviolet detection at 229 nm. For the 0.2 mg/mL formulation, samples were prepared by dissolving scopolamine hydrobromide trihydrate in deionized water and diluting to volume. Aliquots (7 mL) were mixed with internal standard solution (0.04 mg/mL propiophenone in acetonitrile) and were analyzed by HPLC with the same system used to analyze the 11 mg/mL formulation but with a mobile phase ratio of 75:25. The stability of the dose formulations was confirmed for at least 3 weeks at room temperature when stored in the dark or for at least 3 hours at room temperature and open to air and light.Periodic analyses of the dose formulations of scopolamine hydrobromide trihydrate were conducted at the study laboratory using ultraviolet/visible spectrometry for the 14-week studies. During the 14-week studies, dose formulations were analyzed every 6 to 8 weeks. All of the dose formulations analyzed during the 14-week studies were within 10% of the target concentration. For the 14-week , results of periodic referee analyses performed by the analytical chemistry laboratory indicated good agreement with the results of the study laboratory.
Duration of treatment / exposure:
14 weeks
Frequency of treatment:
once daily, 5 days per week
Doses / concentrations
Doses / Concentrations:0, 15, 45, 135, 400, or 1,200 mg/kg bw
No. of animals per sex per dose:
Control animals:
Details on study design:
- Sentinel program: The Sentinel Animal Program is part of the periodic monitoring of animal health that occurs during the toxicologic evaluation of chemical compounds. Under this program, the disease state of the rodents is monitored via serology on sera from extra (sentinel) animals in the study rooms. These animals and the study animals are all subject to identical environmental conditions. The sentinel animals come from the same production source and weanling groups as the animals used for the studies of chemical compounds.


Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes - Time schedule: twice daily
BODY WEIGHT: Yes - Time schedule for examinations: initially, weekly, and at the end of the study
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): feed conumption were recorded weekly by cage
HAEMATOLOGY: Yes - Time schedule for collection of blood: at the end of the study- Anaesthetic used for blood collection: Yes carbon dioxide- Parameters checked which were examined: hematocrit; hemoglobin; erythrocyte, reticulocyte, and nucleated erythrocyte counts; mean cell volume; mean cell hemoglobin; mean cell hemoglobin concentration; platelet count; and leukocyte count and differentials.
OTHER:SPERM MORPHOLOGY AND VAGINAL CYTOLOGY: Yes; From rats receiving 0, 45, 135, 400 mg/kg- The parameters evaluated in males were sperm count, morphology, and motility.The right cauda, right epididymis, and right testis were weighed. Vaginal fluid samples were collected for up to 7 consecutive days prior to the end of the studies for vaginal cytology evaluations. The parameters evaluated in females were relative frequency of estrous stages and estrous cycle length.
NECROPSY: Yes- All animals were necropsied. Organs weighed included the brain, heart, right kidney, liver, lungs, right ovary, right testis, thymus and uterus.
Sacrifice and pathology:
HISTOPATHOLOGY/GROSS PATHOLOGY: Yes; Complete histopathologic examinations were performed on all control rats, male rats receiving 135 mg/kg or higher, female rats receiving 400 mg/kg or higher. In addition to gross lesions, tissue masses and associated lymph nodes, the tissues examined included: adrenal gland, bone and marrow, brain, clitoral gland, esophagus, heart, kidney, large intestine (cecum, colon, and rectum), small intestine (duodenum, jejunum, and ileum), liver, lung, lymph nodes (mandibular and mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin, spleen, stomach (forestomach and glandular stomach), testis with epididymis and seminal vesicle, thymus, thyroid gland, trachea, urinary bladder, and uterus.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical findings included bilateral pupillary dilation in all dosed males and females and reddening of the eyes in 15 mg/kg males and 135, 400, and 1200 mg/kg males and females. Hyperactivity was visually observed in a few dosed males and females (males: 0 mg/kg, 0/10; 15 mg/kg, 0/10; 45 mg/kg, 1/10; 135 mg/kg, 1/10; 400 mg/kg, 0/10; 1200 mg/kg, 1/10; females: 0/10, 2/10, 5/10, 3/10, 1/10, 1/10). Hypoactivity was also visually observed in some other dosed males and females (males: 0/10, 0/10, 0/10, 0/10, 3/10, 3/10; females: 0/10, 0/10, 0/10, 1/10, 1/10, 5/10) and increased with increasing dose.
mortality observed, treatment-related
Description (incidence):
One female receiving 45 mg/kg, two males and one female receiving 135 mg/kg, six males and one female receiving 400 mg/kg, and eight males and seven females receiving 1200 mg/kg died during the study (Table 1).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The final mean body weights and mean body weight gains of all dosed males and females were significantly lower than those of the control groups.The mortality and lower body weights of the dosed animals suggest that the dosed rats did not eat or drink as much as the controls and some dehydration would have occurred.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The hematology data for rats in the 14 weeks study are listed in Table 2. Hematocrit, hemoglobin concentration, and/or erythrocyte count in male and female rats receiving 45 mg/kg or greater were slightly higher than those of the control groups. In general, these differences were most prominent in the 400 and 1200 mg/kg groups. Higher hematocrit, hemoglobin concentration, and erythrocyte count would be consistent with the hemoconcentration associated with dehydration (relative erythrocytosis). The mortality and lower body weights of the dosed animals suggest that the dosed rats did not eat or drink as much as the controls and some dehydration would have occurred. A minimal to mild mature neutrophilia, evidenced by higher segmented neutrophil numbers than those in the control group, occurred in all dosed male rats. Neutrophilia is often a result of an increased tissue demand for granulocytes due to inflammation. There was, however, no microscopic evidence of inflammation that could account for the neutrophilia. Thus, other mechanisms that alter granulopoiesis and/or rate of release from the bone marrow, redistribution of neutrophil life span could be considered. Other hematology differences were sporadic and were not treatment related.
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The absolute and relative liver weights of 1200 mg/kg females were significantly greater than those of the control group. The absolute and relative thymus weights of 15, 45, 135, and 400 mg/kg males and females and 1200 mg/kg females were significantly lower than those of the control groups (Table 3).
Gross pathological findings:
no effects observed
Description (incidence and severity):
no adverse effects
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
no adverse effects
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
no adverse effects
Other effects:
no effects observed
Description (incidence and severity):
Sperm morphology and vaginal cytology parameters in dosed rats were similar to those in the control groups.
Details on results:
The mortality and lower body weights of the dosed animals suggest that the dosed rats did not eat or drink as much as the controls and some dehydration would have occurred. Some males (0/10, 0/10, 0/10, 2/10, 3/10, 4/10) and females (0/10, 0/10, 1/10, 1/10, 0/10, 3/10) died from esophageal obstructions consisting of feed and bedding material in the posterior pharynx. Tracheal obstruction occurred concurrently with esophageal obstruction as a result of food build-up in the oropharyngeal region. This condition is considered to be secondary to the inhibitory effects of scopolamine hydrobromide trihydrate on salivary gland secretions and on esophageal smooth muscle involved in swallowing. There were no other significant treatment-related gross or microscopic findings.

Effect levels

open allclose all
Key result
Dose descriptor:
Effect level:
45 other: mg/kg bw
Based on:
test mat.
Basis for effect level:
Remarks on result:
other: dosage 5 days a week converted in 32.14 mg/kg bw/day
Dose descriptor:
Effect level:
400 other: mg/kg bw
Based on:
test mat.
Basis for effect level:
Remarks on result:
other: dosage 5 days a week

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1: Survival and Body Weights of Rats in the 14-Week Gavage Study of Scopolamine Hydrobromide Trihydrate

     Mean Body Weight°° (g)    
 Dose (mg/kg)  Survival°  Initial  Final  Change  Final Weight Relative to Controls (%)
 0  10/10  116 +-3  341 +-6 255 +-5   
 15 10/10   116 +-3  312 +-7** 197 +-6**   92
 45 10/10   115 +-3  302 +-4** 188 +-5**   89
 135  8/10°°°  114 +-3  300*-7** 186 +-5**   88
 400 4/10°°°°  115 +-2   304 +-3**  188 +-7**  89
 1,200  2/10 °°°°° 116 +-2   277 +-37**  163 +-30**  81
 0  10/10 96 +-1  203 +-2   107 +-2  
 15  10/10  97 +-1  193 +-2**  96 +-2**  95
 45  9/10°°°°°° 98 +-2   196 +-2*  97 +-1**  97
 135 9/10°°°°°°°  95 +-1   190 +-2**  96 +-3** 94 
 400  9/10°°°°°° 96 +-1   189 +-2**  93 +-3** 93 
 1200  3/10°°°°°°°° 95 +-1  192 +-6**   95 +-5**  94

* Significantly different(P<=0.05) from the control group by Williams’ or Dunnett’s test

** P<=0.01

°Number of animals survivinglnumber initially in group. Subsequent calculations are based on animals surviving to the end of the study

°°Weights and weight changes are given as mean f standard error

°°°Week of death: 7, 14

°°°°Week of death: 1, 2,7, 7,13, 13

°°°°°Week of death: 1, 1, 1, 3, 4, 5, 9, 14

°°°°°°Week of death: 4

°°°°°°°Week of death: 8

°°°°°°°°Week of death: 1, 2,5, 6, 7, 7

Table 2: Hematology Data for Rats in the 14-WeekGavage Study of Scopolamine Hydrobromide Trihydrate°

   Vehicle Control 15 mg/kg  45 mg/kg  135 mg/kg  400 rng/kg  1,200 mg/kg 
10   10 1°° 
Hematocrit (%)  46.8+-0.5  46.2+-0.4   48.0+-0.3* 48.1+-0.3*   49.0+-0.4**  48.9
Hemoglobin (g/dL)  15.2+-0.2  15.3+-0.2  15.7+-0.1   15.9 f 0.1*  16.2+-0.1** 16.4
Erythroctes (10^6/µL)   9.39+-0.10  9.38+-0.10  9.63+-0.08 9.61 f 0.05 9.72+-0.11   9.58
Reticulocytes (10^6/µL)  0.50+-0.03  0.47+-0.02  0.52+-0.03  0.42 f 0.03  0.40+-0.04  0.46 
Nucleated erythrocytes (10^3/µL)  0.03+-0.01  0.04+-0.01  0.01+-0.01   0.02+-0.01  0.02+-0.02 0.08 
Mean cell volume (fL)  49.9+-0.4   49.1+-0.1  50.0+-0.2 50.0+-0.3  50.5+-0.3  51.0 
Mean cell hemoglobin (pg) 16.2+-0.3  16.3+-0.1   16.3+-0.1 16.6+-0.1   16.7+-0.1 17 .1 
Mean cell hemoglobin concentration (g/dL) 32.6+-0.5  33.2+-0.1  32.8+-0.2   33.1+-0.2 33.1+-0.1   33.5
Platelets ( 10^3/µL) 633.4+-20.8  638.8+10.6  655.8+-28.2°°°  620.6+-20.6  621.5+-21.3  601.0 
Leukocytes (10^3/µL)  7.35+-0.44  6.96+-0.31   8.01+-0.36 7.59+-0.56   6.73+-0.57 8.40 
Segmented neutrophils (10^3/µL) 1.14+-0.13  1.89+-0.28*  2.07+-0.17**   1.82+-0.20** 1.90+-0.12*  2.69 
Lymphocytes (10^3/µL)  5.65+-0.35  4.68+-0.34  5.67+-0.41  5.37+-0.37   4.65+-0.45 5.38 
Atypical lymphocytes (10^3/µL) 0.12+-0.03  0.08+-0.03  0.04+-0.02  0.08+-0.05  0.00+-0.00  0.00 
Monocytes (10^3/µL)  0.35+-0.09  0.22+-0.06  0.17+-0.05  0.20+-0.04   0.07+-0.04* 0.34 
 Eosinophils (10^3/µL) 0.08+-0.03   0.09+-0.03 0.06+-0.01  0.11+-0.04   0.08+-0.02  0.00

   Vehicle Control 15 mg/kg  45 mg/kg  135 mg/kg  400 rng/kg  1,200 mg/kg 
10   9
Hematocrit (%)  46.9+-0.4  47.4+-0.6   48.1+-0.4 47.6+-0.4   49.0+-0.4**  49.1+-0.9*
Hemoglobin (g/dL)   15.6+-0.1  15.5+-0.2 15.9+-0.1  15.7+-0.2  16.2+-0.1**  16.4+-0.3*
Erythroctes (10^6/µL)  8.82+-0.07   8.88+-0.11  9.09+-0.07*  8.98+-0.10  9.17+-0.08*  9.26+-0.17*
Reticulocytes (10^6/µL)  0.41+-0.02  0.38+-0.02  0.42+-0.03  0.43+-0.03  0.43+-0.03  0.42+-0.05 
Nucleated erythrocytes (10^3/µL)  0.02+-0.01  0.05+-0.01   0.03+-0.02 0.05+-0.02  0.05+-0.02  0.07+-0.07 
Mean cell volume (fL)   53.1+-0.3 53.2+-0.2   52.7+-0.2 53.1+-0.2   53.5+-0.3 53.0+-0.0 
Mean cell hemoglobin (pg) 17.7+-0.1  17.4+-0.1  17.5+-0.1   17.5+-0.1  17.7+-0.1 17.7+-0.1 
Mean cell hemoglobin concentration (g/dL) 33.3+-0.2  32.6+-0.1*   33.1+-0.1 33.0+-0.2   33.1+-0.1 33.5+-0.2 
Platelets ( 10^3/µL) 759.6+-39.6  764.3+-31.7  758.4+-27.5   807.4+-29.8 810.1+-44.3  698.7+-14.6 
Leukocytes (10^3/µL)  6.37+-0.33   6.23+-0.40 6.21+-0.43  8.57+-0.38*  7.39+-0.59  5.97+-0.91 
Segmented neutrophils (10^3/µL) 1.45+-0.11  1.30+-0.16  1.45+-0.12  2.16+-0.29  1.73+-0.17   1.68+-0.57
Lymphocytes (10^3/µL)  4.63+-0.36   4.68+-0.25 4.48+-0.42   6.15+-0.25* 5.35+-0.48  4.10+-0.38 
Atypical lymphocytes (10^3/µL) 0.09+-0.04  0.03+-0.03   0.02+-0.01  0.04+-0.04 0.04+-0.03  0.04+-0.02 
Monocytes (10^3/µL)  0.14+-0.05  0.15+-0.04  0.18+-0.06  0.15+-0.07  0.20+-0.08  0.08+-0.03 
 Eosinophils (10^3/µL) 0.06+-0.01  0.07+-0.02  0.07+-0.02   0.07+-0.03 0.04+-0.01  0.07+-0.04 

* Significantly different (P<=0.05) from the control group by Dunn’s or Shirley’s test

** P<=0.01

° Mean f standarderror.Statisticaltests wereperformedonunroundeddata.

°°n=l; no standard error calculated


Applicant's summary and conclusion

Under the test conditions a NOAEL of 45 mg/kg bw (male) and a NOAEL of 400 mg/kg bw (female) was determined.