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Toxicological information

Carcinogenicity

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Description of key information

The most sensitive chronic NOAEL value of 5 mg/kg bw (five days a week) was determined in a NTP (National Toxicology Program) study conducted with rats for 2 years (oral gavage) with Scopolamine hydrobromide trihydrate. The exposure for five days per week under test conditions was extrapolated to seven days per week resulting in the NOAEL value of 3.57 mg/kg bw/day. As the study was conducted with the read across substance Scopolamine hydrobromide trihydrate a molecular weight conversion was done. After molecular weight conversion a NOAEL value of 2.46 mg/kg bw/day could be used for DENEL calculation.

No evidence of carcinogenic activity for the substance was found in the study, the NOAEL value is based on survival.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records
Reference
Endpoint:
carcinogenicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Deviations:
not specified
GLP compliance:
yes
Remarks:
Food and Drug Administration (FDA) Good Laboratory Practice Regulations (21 CFR, Part 58)
Species:
rat
Strain:
other: F-344/N
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Simonsen Laboratories (Gilroy, CA)
- Age at study initiation: approximately 6 weeks
- Housing: five per cage; Cages: Polycarbonate (Lab Products Inc., Maywood, NJ), changed twice weekly; Bedding: Sani-Chip® hardwood chips (P.J. Murphy Forest Products Corp., Montville, NJ); Cage filters: DuPont 2024 spun-bonded polyester (Snow Filtration Co., Cincinnati, OH); Racks: Stainless steel (Lab Products Inc., Maywood, NJ), rotated once every 2 weeks
- Diet (e.g. ad libitum): NIH-07 open formula pellet diet (Zeigler Brothers, Inc., Gardners, PA) available ad libitum
- Water (e.g. ad libitum): tap water (Columbus Municipal Supply) via automatic watering system (Edstrom Industries, Waterford, WI) available ad libitum
- Acclimation period: 14 days quarantinedENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.7 to 23.3- Humidity (%): 24 to 72
- Air changes (per hr): minimum of 10 changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours/day fluorescent light
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
deionized
Analytical verification of doses or concentrations:
yes
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 HPLC for the 2-year studies. During 2-year studies, dose formulations were analyzed every 6 to 8 weeks. During the 2-year studies, 92% (72/78) of the dose formulations were within 10% of the target concentration with no value differing more than 20% from the target concentration. For the 2-year studies, 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:
104 weeks
Frequency of treatment:
once daily, 5 days per week
Remarks:
Doses / Concentrations: 0, 1, 5, 25 mg/kg body weight Basis: actual ingested
No. of animals per sex per dose:
60
Control animals:
yes
yes, historical
Details on study design:
- Dose selection rationale: Based on the lower mean body weights in all dosed animals and lower survival in 400 mg/kg males and 1200 mg/kg males and females compared to body weights and survival in the control groups in the 14 weeks in rat study, doses selected for the 2 years study were 1, 5, and 25 mg/kg.
Observations and examinations performed and frequency:
ANIMAL OBSERVATIONS: Yes - Time schedule: twice daily
DETAILED CLINICAL OBSERVATIONS: Yes - Time schedule: every 3-4 weeks during the first 13 weeks and monthly thereafter
BODY WEIGHT: Yes - Time schedule for examinations: weekly through the first 13 weeks, monthly thereafter, and at the end of the study
OPHTHALMOSCOPIC EXAMINATION: Yes- Time schedule for examinations: Before the study began and at 15 months- Dose groups that were examined: 10 male and 10 female per group
HAEMATOLOGY: Yes - Time schedule for collection of blood: At the 15-month interim evaluation (within the hour after dosing)
- Anaesthetic used for blood collection: Yes (CO2:O2))
- How many animals: 10 male and 10 female rats per group (excluding female rats that received 1 mg/kg*)
- Parameters 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.* Because of a flooding incident that killed 16 female rats receiving 1 mg/kg, no females in this dose group were bled or necropsied at 15 months.
CLINICAL CHEMISTRY: No
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: Yes- Time schedule for examinations: Prior to the study, on the first day of study, and after 3, 6, 9, 12, and 24 months of exposure.
- Dose groups that were examined: Ten male and ten female rats per dose group. The same animals were used at each time point.
- Battery of functions tested: motor activity, grip strength, thermal sensitivity, startle responsiveness, and passive avoidance.
NECROPSY: Yes- All core-study animals were necropsied. Organs weighed at the 15-month interim evaluations were the right epididymis, right kidney, liver, right testis (excluding female rats receiving 1 mg/kg). At necropsy, all organs and tissues were examined for gross visible lesions.
PLASMA SCOPOLAMINE DETERMINATION: Yes- At 15 months, 10 male and 10 female rats were bled for plasma scopolamine determination. (Rats had been bled one hour earlier for hematology evaluations.)
Sacrifice and pathology:
HISTOPATHOLOGY/GROSS PATHOLOGY: Yes; Complete histopathologic examinations were performed on all core-study animals. 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 or mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, spleen, stomach (forestomach and glandular stomach), testis with epididymis and seminal vesicle, thymus, thyroid gland, trachea, urinary bladder, and uterus.
Statistics:
The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958).Statistical analyses for possible dose-related effects on survival used Cox's (1972) method for testing two groups for equality and Tarone's (1975) life table test to identify dose-related trends. All reported P values for the survival analyses are two sided.For calculation of statistical significance, the incidences of most neoplasms and all non-neoplastic lesions are given as the ratio of the number of affected animals to the number of animals with the site examined microscopically.Tests: prevalence analysis of Dinse and Lagakos (1983), further described and illustrated by Dinse and Haseman (1986).Other methods: the life table test (Cox, 1972; Tarone, 1975), appropriate for rapidly lethal neoplasms, the Fisher exact test and the Cochran-Armitage trend test (Armitage, 1971; Gart et al., 1979)Organ and body weight data (approximately normal distribution) were analyzed using the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972).Hematology and epididymal spermatozoal data (typically skewed distribution), were analyzed using the nonparametric multiple comparison methods of Shirley (1977) and Dunn (1964).Jonckheere’s test (Jonckheere, 1954) was used to assess the significance of the dose-related trends and to determine whether a trend-sensitive test (Williams’ or Shirley’s test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose-related trend (Dunnett’s or Dunn’s test). Prior to analysis, extreme values identified by the outlier test of Dixon and Massey (1951) were eliminated.Average severity values were analyzed for significance using the Mann-Whitney U test (Hollander and Wolfe, 1973).Because vaginal cytology data are proportions, an arcsine transformation was used. Treatment effects were investigated by applying a multivariate analysis of variance (Morrison, 1976).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical findings included bilateral pupillary dilation in all dosed males and females. Ophthalmic examination revealed no significant findings.
Mortality:
mortality observed, treatment-related
Description (incidence):
The survival of female rats receiving 1 and 25 mg/kg were significantly lower than that of the control group. The lower survival in 1 mg/kg females was due primarily to a cage flooding accident resulting from a malfunctioning automatic watering system valve
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean body weights of 1 and 5 mg/kg males and females were similar to those of the controls throughout the study. The mean body weight of 25 mg/kg males was slightly lower than that of the control group from about week 25 through week 97. The mean body weight of 25 mg/kg females was lower than that of the control group from week 25, and the final mean body weight was 81% that of the controls.
No adverse effects observed.
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:
no effects observed
Description (incidence and severity):
No adverse effects.
Haematological findings:
no effects observed
Description (incidence and severity):
Compared to controls, hematocrit was slightly higher in the 25 mg/kg male rats, similar to the effects observed in the 14-week study; this would be consistent with dehydration resulting in hemoconcentration. The reticulocyte count in 25 mg/kg female rats was slightly lower than that in the control group. This result is consistent with the lower body weights, and thus a decreased nutritional status, exhibited by these animals.
No adverse effects.
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No adverse effects.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No adverse effects (See Table 2)
Gross pathological findings:
no effects observed
Description (incidence and severity):
The incidences of adenoma of the pituitary gland pars distalis decreased with increasing dose in both male and female rats; however, this trend was only significant in males (males: vehicle control, 19/49; 1 mg/kg, 17/49; 5 mg/kg, 13/50; 25 mg/kg, 10/50; females: 20/50, 13/60, 14/50, 10/50). The incidences of adenoma of the pituitary gland pars distalis in 25 mg/kg males and all groups dosed females were below the NTP historical control range. The incidences of hyperplasia were not significantly different from those in the control groups.The incidences of mononuclear cell leukemia in 25 mg/kg males and females were significantly lower than those of the control groups (males: 33/50, 21/50, 26/50; females: 20/50, 6/60, 13/50, 4/50). The incidence of mononuclear cell leukemia in females receiving 25 mg/kg was well below the NTP historical range
No adverse effects observed.
Neuropathological findings:
effects observed, treatment-related
Description (incidence and severity):
Horizontal motor activity of 25 mg/kg females was significantly greater than that of the control group on days 90, 180, and 360. The startle response of 5 and 25 mg/kg females was significantly lower than that of the control group on day 90. On day 180, passive avoidance by 25 mg/kg males was significantly lower than that by the control group.
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.
Description (incidence and severity):
Plasma Scopolamine Determinations:Serum samples collected from rats 1 hour after dosing were analyzed to determine serum scopolamine concentrations. Serum scopolamine concentration analyses indicated 6 ng scopolamine/mL serum for the 5 mg/kg female sample and 12 and 28 ng/mL for the 25 mg/kg male and female samples, respectively. The amounts of scopolamine in the other serum samples were below the minimum detection limit (4 ng/mL) of the analysis method.
Key result
Dose descriptor:
NOAEL
Effect level:
5 other: mg/kg bw
Based on:
test mat.
Sex:
female
Basis for effect level:
mortality
Remarks on result:
other: dosage 5 days a week converted in 3.57 mg/kg bw/day
Dose descriptor:
NOAEL
Effect level:
>= 25 other: mg/kg bw
Based on:
test mat.
Sex:
male
Basis for effect level:
mortality
Remarks on result:
other: dosage 5 days a week
Critical effects observed:
not specified

Table 1: Summary of the 2 -Year Carcinogenesis Toxicology Study of Scopolamine Hydrobromide Trihydrate

   Male F344/N Rats  Female F344/N Rats
 Doses  0, 1, 5, 25 mg/kg in water by gavage   0, 1, 5, 25 mg/kg in water by gavage
 Body weights  25 mg/kg group lower than control group    25 mg/kg group lower than control group
 2 -Year survival rates  20/50, 14/50, 22/50, 28/50  34/50, 17*/60, 26/50, 22/50
 Nonneoplastic effects  None  None
 Neoplastic effects  None  None
 Levels of evidence of carcinogenic activity  No evidence  No evidence

*The lower survival in 1 mg/kg females was due primarily to a cage flooding accident resulting froma malfunctioning automatic watering system valve.

Table 2: Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats at the 15-Month Interim Evaluation in the 2-Year Gavage Study of Scopolamine Hydrobromide Trihydrate

   Vehicle Control  1 mg/kg  5 mg/kg  25 mg/kg
 Male        
 n  10  10  10  10
 Necropsy body wt  493 +- 9 492 +- 11   481 +- 10  448 +- 9**
 R. Epididymis        
 Absolute  0.423 +- 0.022  0.464 +- 0.012  0.428 +- 0.014 0.441 +- 0.017 
 Relative  0.86 +- 0.04  0.94 +- 0.02 0.89 +- 0.03   0.99 +- 0.04*
 R. Kidney        
 Absolute  1.614 +- 0.029  1.565 +- 0.050  1.543 +- 0.047  1.366 +- 0.036**
 Relative 3.28 +- 0.07   3.18 +- 0.06  3.20 +- 0.06  3.06 +- 0.08
 Liver        
 Absolute  18.032 +- 0.477  17.953 +- 0.579  17.382 +- 0.580  15.319 +- 0.481**
 Relative  36.65 +- 0.83  36.46 +- 0.71  36.11 +- 0.81  34.21 +- 0.84
 R. Testis        
 Absolute  1.884 +- 0.229  1.680 +- 0.075  1.777 +- 0.169  1.669 +- 0.142
 Relative  3.83 +- 0.47  3.43 +- 0.18  3.67 +- 0.30  3.76 +- 0.37

 Female        
 n  10    10  10
 Necropsy body wt  293 +- 12  -b)  285 +- 5  274 +- 7
 R. Kidney        
 Absolute  0.938 +- 0.017  -  0.869 +- 0.014**  0.845 +- 0.021**
 Relative 3.23 +- 0.10   -  3.06 +- 0.07  3.09 +- 0.04
 Liver        
 Absolute  9.198 +- 0.322  -  8.869 +- 0.147  8.392 +- 0.235*
 Relative  31.54 +- 0.92  -  31.19 +- 0.43  30.70 +- 0.59
 * Significantly +- (P<=0.05) from the control group by Williams' or Dunnett's test** P<=0.01a) Organ weights and body weights are given in grams; organ-weight-to-body-weight ratios are given as mg organ weight/g body weight (mean +- standard error).b) Female rats in the 1 mg/kg group wen not necropsied at the interim evaluation due to high mortality.
Conclusions:
Under the test conditions a NOAEL of >=25 mg/kg bw (male) and a NOAEL of 5 mg/kg bw (female) was determined. The exposure for five days per week under test conditions was extrapolated to seven days per week resulting in the NOAEL value of 3.57 mg/kg bw/day for female.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
2.46 mg/kg bw/day
Study duration:
chronic
Species:
rat

Carcinogenicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Carcinogenicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A 2 -year carcinogenicity study (NTP) was conducted in accordance with good scientific principles with the read across substance Scopolamine hydrobromide trihydrate. Under the test conditions a NOAEL of >= 25 mg/kg bw (male) and a NOAEL of 5 mg/kg bw (female) was determined for rats (F344/N) and a NOAEL of >= 25 mg/kg bw (female/male) was determined for mice (B6C3F1).The exposure for five days per week under test conditions was extrapolated to seven days per week resulting in the NOAEL value of 3.57 mg/kg bw/day. As the study was conducted with the read across substance Scopolamine Hydrobromide Trihydratea molecular weight conversion was done. After molecular weight conversion the NOAEL value 2.46 mg/kg bw/day could be used for DENEL calculation.

Under the condition of these 2 -year gavage studies, there was no evidence of carcinogenic activity of the read across substance scopolamine hydrobromide trihydrate in male or female rats or mice administered 1, 5, or 25 mg/kg. All NOAEL values are based on survival.

Justification for classification or non-classification

Based on the available data, the substance will not be classified.