Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In an in vitro gene mutation test (key in vitro Ames test, NTP 1997) conducted with the read-across substance Scopolamine hydrobromide trihydrate, no adverse effects were observed (negative). This result will be supported by a Klimisch scored 4 Ames test (supporting in vitro Ames test, Pares 2003) conducted with the target substance Scopolamine.

In an in vitro cytogenicity test in mammalian cells (key in vitro chromosome aberration test, NTP 1997), adverse effects were observed at the highest concentration tested (5000 mg/L) with metabolic activation. This result will be supported by a Klimisch scored 4 in vitro chromosome abberation and sister chromatid exchange test (supporting, Yu 1988). However, no adverse effects were observed for the read-across substance in an in vivo micronucleus assay in mice (key in vivo micronucleus test, NTP 1997). In addition the carcinogenic potential of scopolamine hydrobromide trihydrate was investigated in two-year gavage studies in mice and rats(section 5.8 of the CSR). There was no evidence of carcinogenic activity of scopolamine hydrobromide trihydrate in male or female B6C3F1 mice or F344/N rats administered at doses of 1, 5, or 25 mg/kg/day.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 March 1997
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
-S9: 1000, 1600, 3000 µg/mL
+S9: 1600, 3000, 5000 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
DURATION
- Exposure duration:
- without S9: treatment time 10.5 hr; when after the addition of Colcemid: incubation time 2 hr
- with S9: treatment time 2 hr; when change of medium; incubation time 11 to 11.5 hr with Colcemid present in the final 2 hr
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): GiemsaIn the aberration test without S9, cells were incubated in McCoy's 5A medium with scopolamine hydrobromide trihydrate for 10.5 hours; Colcemid was added and incubation continued for 2 hours. The cells were then harvested by mitotic shake-off, fixed, and stained with Giemsa. For the Aberration with S9, cells were treated with scopolamine hydrobromide trihydrate and S9 for 2 hours, after which the treatment medium was removed and the cells were incubated for 11 to 11.5 hours in fresh medium, with Colcemid present for the final 2 hours. Cells were harvested in the same manner as for the treatment without S9. As an alteration in pH was observed in the first trial conducted with S9 and the second trial with S9 was conducted with HEPES buffer present in the medium to stabilize pH.
NUMBER OF CELLS EVALUATED: 200 first-division metaphase cells were scored at each dose level
Statistics:
To arrive at a statistical call for a trial, analyses were conducted on both the dose response curve and individual dose points. For a single trial, a statistically significant (P<=0.05) difference for one dose point and a significant trend (P<=0.015) were considered weak evidence for a positive response; significant differences for two or more doses indicated the trial was positive. A positive trend test in the absence of a statistically significant increase at any one dose resulted in an equivocal call.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
the high dose was limited by toxicity in the trials conducted without S9
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
with S9, no toxicity was noted and 5 mg/mL was selected as the high dose
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Induction of Chromosomal Aberration in Chinese Hamster Ovary Cells by Scopolaminehydrobromide Trihydrate

 

   Dose (µg/mL)  Total Cells  No. of Abs  Abs/Cell  Cells with Abs (%)    Dose (µg/mL)  Total Cells  No. of Abs  Abs/Cell  Cell with Abs (%)
  -S9                              +S9
 DMSO    200  2  0.01  1.0 DMSO     200  1  0.01  0.5
 Mitomycin C  0.0625  200  40  0.20  16.0  Cyclo- phosphamide  2.5  200  35  0.18  15.0
   0.2500  50  17  0.34  30.0    7.5  50  23  0.46  36.0
 Scopolamine-hydrobromidetrihydrate  1000  200  1  0.01  0.5  Scopolamine-hydrobromidetrihydrate(Trial 1)  1600  200  3  0.02  1.5
   1600  200  2  0.01  1.0    3000  200  2  0.01  1.0
   3000  200  1  0.01  0.5    5000  200  12  0.06  6.0
           P=0.655            P<0.001
             (Trial 2) DMSO    200  1  0.01  0.5
             Cyclophosphamide  2.5  200  28  0.14  12.0
               7.5  50  17  0.34  34
             Scopolamine-hydrobromide trihydrate (Trial 2)  1600  200  3  0.02  1.5
               3000  200  2  0.01  1.0
               5000  200  28  0.14  11.0
                       P<0.001
Conclusions:
Positive with metabolic activation. Induction of chromosome aberration was observed in cultured CHO cells in the presence of S9 metabolic activation in each of two trials at the highest dose tested (5000 µg/mL).
Executive summary:

The present study determined the mutagenicity of Scopolamine hydrobomide trihydrate according to a well-described method which is similar to a current internationally established guidelines (OECD 473). The effects were analysed using the Chinese hamster ovary cells with or without S9 mix (from Sprague Dawley rat liver). The study was part of the National Toxicology Program (NTP) of the U.S. Department of Health and Human Services and reviewed and showed that the test item has mutagenic properties under conditions of metabolic activation.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 March 1997
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
Strain; allele; DNA targetTA1535; hisG46; GGGTA1537; hisC3076; +1 frameshift and CCCTA98; hisD3052; CGCGCGCGTA100; hisG46; GGGTA97; hisD6610; CCCCCC
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 97
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
0, 100, 333, 1000, 3333, 10000 µg test substance/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 4-nitro-o-phenylenediamine; 2-aminoanthracene
Remarks:
without metabolic activation: sodium azide (TA1535 and TA100), 9-aminoacridine (TA97 and TA1537), 4-nitro-o-phenylenediamine (TA98) with metabolic activation: 2-aminoanthracene (all strains)
Evaluation criteria:
A positive response is defined as a reproducible, dose related increase in histidine-independent (revertant) colonies in any one strain/activation combination. An equivocal response is defined as an increase in revertants that is not dose related, is not reproducible, or is of insufficient magnitude to support a determination of mutagenicity. A negative response is obtained when no increase in revertant colonies is observed following chemical treatment. There was no minimum percentage of fold increase required for a chemical to be judged positive or weakly positive.
Statistics:
Not regarded as necessary according to the OECD guidelines.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Mutagenicity of Scopolamine Hydrobromide Trihydrate in Salmonella typhimurium. Revertants are presented as mean +- standard error from three plates

 Strain     Dose (µg/plate)     -S9        +hamster S9     +rat S9
 Trial 1  Trial 2  10%  30%  10%  30%
 TA100  0  102 +-6.4  102 +-7.8  87 +-8.1  101 +-4.8  101 +-9.0  108 +-8.4
   100  117 +-17.5  95 +-8.8  90 +-2.6  106 +-5.8  90 +-3.2  106 +-4.7
   333  100 +-4.6  94 +-2.3  101 +-7.5  105 +-4.6  91 +-3.6  105 +-3.3
   1000  87 +-6.1  108 +-2.4  90 +-7.4  107 +-7.2  88c  86 +-2.7
   3333  90 +-10.0  96 +-4.3  98 +-7.3  122 +-6.7  94 +-8.5  113 +-4.4
   10000  98 +-34.0  94 +-8.0  85 +-5.1  97 +-2.3  83 +-2.6  107 +-4.7
 Trial summary    negative  negative  negative  negative  negative  negative
 positive control    281 +-11.8  569 +-21.2  283 +-11.6  426 +-4.7  309 +-1.5  294 +-9.1
               
 TA 1535  0  21 +-1.3  27 +-0.3  11 +-0.7  17 +-2.1  9 +-3.5  15 +-2.3
   100  19 +-2.6  35 +-4.2  13 +-3.5  12 +-2.0  9 +-1.2  13 +-1.5
   333  21 +-4.8  33 +-2.3  12 +-2.3  11 +-0.9  11 +-0.9  12 +-1.9
   1000  21 +-5.2  30 +-1.5  10 +-1.7  8 +-0.7  10 +-0.9  12 +-1.5
   3333  28 +-1.5  29 +-6.0  13 +-1.3  13 +-0.7  14 +-2.2  12 +-1.9
   10000  23 +-1.9  36 +-6.0  9 +-0.7  9 +-1.5  12 +-1.3  12 +-0.3
 Trial summary    negative  negative  negative  negative  negative  negative
 positive control    307 +-8.2  369 +-6.7  68 +-4.5  96 +-12.4  202 +-9.5  69 +-0.5
               
 TA1537  0  6 +-1.2  19 +-2.7  21 +-2.3  11 +-1.5  21 +-2.8  8 +-1.0
   100  8 +-2.0  20 +-4.2  18 +-1.7  9 +-1.7  19 +-1.3  5 +-2.2
   333  5 +-0.3  18 +-0.9  23 +-2.0  11 +-2.1  25 +-0.7  6 +-1.5
   1000  4 +-1.8  21 +-2.7  20 +-1.9  10 +-0.7  20 +-2.0  8 +-1.5
   3333  7 +-1.0  20 +-2.9  20 +-2.4  10 +-1.2  21 +-1.2  7 +-1.7
   10000  6 +-2.7  12 +-1.5  21 +-4.4  8 +-2.2e  25 +-2.2  5 +-1.5
 Trial summary    negative  negative  negative  negative  negative  negative
 positive control    825 +-65.6  38 +-5.2  465 +-15.7  72 +-2.9  60 +-11.1  28 +-1.5
               
 TA98  0  14 +-3.0  21 +-2.8  37 +-1.5  31 +-2.2  29 +-3.8  23 +-1.7
   100  11 +-1.0  17 +-3.7  33 +-1.2  33 +-3.5  33 +-1.3  23 +-2.4
   333  18 +-1.7  14 +-0.9  23 +-3.1  28 +-5.2  31 +-4.4  29 +-4.5
   1000  16 +-6.0  12 +-1.5  27 +-0.9  30 +-5.6  30 +-3.8  26 +-3.5
   3333  15 +-10.0  18 +-1.9  30 +-2.5  30 +-2.3  33 +-2.5  29 +-1.7
   10000  14 +-3.5  20 +-3.6  27 +-1.0  28 +-3.6  25 +-5.2  25 +-2.6
 Trial summary    negative  negative  negative  negative  negative  negative
 positive control    114 +-5.2  224 +-13.6  85 +-3.2  74 +-9.7  70 +-4.7  109 +-9.0
               
 TA97  0  79 +-2.6  72 +-5.8  106 +-5.6  200 +-16.6  122 +-11.7  133 +-13.0
   100  79 +-3.0  74 +-7.1  118 +-8.3  211 +-22.3  120 +-7.4  168 +-7.6
   333  74 +-3.4  70 +-8.1  117 +-8.1  172 +-10.7  123 +-5.2  178 +-6.1
   1000  79 +-5.9  71 +-1.5  115 +-6.6  186 +-12.0  114 +-7.8  164 +-11.3
   3333  83 +-3.3  59 +-1.2  106 +-4.0  286 +-21.1  118 +-2.5  133 +-15.0
   10000  84 +-7.8  55 +-2.7  106 +-4.2  266 +-11.3  101 +-2.0  117 +-113
 Trial summary    negative  negative  negative  negative  negative  negative
 Positive control    1302 +-75.0  198 +-16.3  600 +-43.4  952 +-93.1  761 +-42.0  309 +-9.9

C No standard error calculated due to loss of replicate cultures

D The positive controls in the absence of metabolic activation were sodium azide (TA1535 and TA100), 9 -aminoacridine (TA97 and TA1537), and 4-nitrophenylenediamine (TA98). The positive control for metabolic activation with all strains was 2-aminoanthracene.

Conclusions:
Scopolamine hydrobromide trihydrate (100 to 10000 µg/plate) did not induce mutations in Salmonella typhimurium strains TA97, TA98, TA100, TA1535 or TA1537 with or without S9 mix (from Sprague Dawley rat or Syrian hamster liver).
Executive summary:

The present study determined the mutagenicity of Scopolamine hydrobomide trihydrate according to a well-described method which is similar to a current internationally established guideline (OECD 471). The effects were analysed using the Salmonella typhimurium strains TA97, TA98, TA100, TA1535 or TA1537 with or without S9 mix (from Sprague Dawley rat or Syrian hamster liver). However, a bacterial strain for the analysis of oxidizing mutagens, cross-linking agents and hydrazines was not included in testing. The study was part of the National Toxicology Program (NTP) of the U.S. Department of Health and Human Services and reviewed and showed that the test item is devoid of mutagenic properties.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

One in vivo micronucleus assay in mice (key in vivo micronucleus test, NTP 1997) for the read-across substance Scopolamine hydrobromide trihydrate is available. No adverse effects (negative) were observed in this test for the read-across substance.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
01 March 1997
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Principles of method if other than guideline:
- repeated exposure over 14 weeks
GLP compliance:
no
Type of assay:
micronucleus assay
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Simonsen Laboratories, Inc. (Gilroy, CA)- Age at study initiation: 6 or 7 weeks
- Housing: mouse were housed individually
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
ENVIRONMENTAL CONDITIONS
- Temperature: 20 to 24.9°C- Humidity: 45% to 71%
- Air changes (per hr): min. 10 changes per hour- Photoperiod (hrs dark / hrs light): 12/12 dark/light
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:0, 15, 45, 135, 400 or 1200 mg scopolamine hydrobromide trihydrate/kg body weight in distilled water by gavage for 14 weeks
Duration of treatment / exposure:
repeated exposure 14 weeks
Frequency of treatment:
repeated exposure 14 weeks
Remarks:
Doses / Concentrations:0, 15, 45, 135, 400 or 1200 mg/kg bwBasis:nominal conc.
No. of animals per sex per dose:
10 male and 10 female mice
Control animals:
yes, concurrent vehicle
Positive control(s):
none
Tissues and cell types examined:
Blood
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Peripheral blood samples were obtained from male and female B6C3F1 mice at the end of the 14 week toxicity study.DETAILS OF SLIDE PREPARATION: Smears were immediately prepared and fixed in absolute methanol, stained with a chromatin- specific fluorescent dye mixture of Hoechst 33258/pyronin Y (MacGregor et al., 1983).METHOD OF ANALYSIS: Slides were scanned at 630 or 1000x magnification with a semi-automated image analysis system to determine the frequency of micronuclei in 10000 normochromatic erythrocytes (NCEs) in each of 10 animals per dose group.
Evaluation criteria:
In the micronucleus test an individual trial was considered positive if the trend test P value was less than or equal to 0.025 or the P value for any single exposure group was less than or equal to 0.025 divided by the number of exposure groups.
Statistics:
Testing for increasing trend over exposure groups: one-tailed Cochran-Armitage trend test, followed by pairwise comparisons between each exposure group and the control group
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
not specified

Frequency of Micronuclei in Mouse Peripheral Blood Erythrocytes Following Treatment with Scopolamine Hydrobromide Trihydrate by Gavage for 14 weeksa

   Dose (mg/kg)  Micronucleated NCEs/1000 Cellsb  Number of Mice
 Male      
   0  1.95 +-0.14  10
   15  2.12 +-0.06  10
   45  2.18 +-0.20  10
   135  2.22 +-0.12  10
   400  1.91 +-0.21  10
   1200  2.24 +-0.21  10
 Trend test    P=0.714c  
 Female      
   0  1.59 +-0.09  10
   15  1.43 +-0.11  10
   45  1.64 +-0.14  10
   135  1.43 +-0.09  10
   400  1.40 +-0.12  10
   1200  1.38 +-0.12  10
 Trend test    P=0.1219  

a The detailed scoring protocol is presented by Mac Gregor et al. (1990); 10000 NCEs scored per animal

b Data presented as mean +- standard error of the mean. NCE = normochromatic erythrocyte

c Significance of micronucleated NCEs determined by a one-tailed Cochran-Armitage trend test

Conclusions:
No increase in the frequency of micronucleated NCEs was noted in peripheral blood samples obtained from male and female mice at the termination of the 14-week toxicity studies with scopolamine hydrobromide trihydrate up to a concentration of 1200 µg/kg bw.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

In-vitro studies

For the both Ames tests, one according to OECD TG 471 (supporting in vitro Ames test, Pares, 2003) using the test target substance and one similar to OECD TG 471 using the read-across substance (key, in vitro Ames test, NTP 1997), no mutagenic effects were determined for the bacterial strains S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 with and without metabolic activation up to 0.025 ml/plaque and 10000 µg/plate, respectively.

However, mutagenic effects were determined for the read-across substance in a reliable chromosomen aberration Test (key in vitro chromosome aberration test, NTP1997) . In this in vitro mammalian chromosome aberration test conducted similar to OECD 473, Chinese hamster ovary cells were analysed. The cells were treated with 1000 to 3000 µg /ml for 10.5 h of exposure time without metabolic activation, and with 1600 to 5000 µg/ml for 2 h exposure time and additional 9 h incubation time with fresh medium with metabolic activation. A biologically, statistically significant and verified (repetition of experiment) increase in the percentage of cells with aberrations was observed at the highest dose with metabolic activation. It was concluded that Scopolamine hydrobomide trihydrate was positive for the induction of structural aberrations in the presence of metabolic activation under this in vitro test conditions.

In-vivo study

In an in vivo micronucleus test using B6C3F1 mice, which was conducted similar to OECD TG 474, ten animals per sex and dose were treated by gavage with 15, 45, 135, 400 and 1200 mg Scopolamine hydrobomide trihydrate per kg bw in a 14 weeks toxicity study. No significant increase in the incident of micronucleated NCE's (normochromatic erythrocytes) was observed in the peripheral blood samples of male or female mice at the end of the 14 weeks toxicity study up to 1200 mg/kg bw. It is concluded that the test substance is negative for the induction of micronuclei under this in vivo conditions of the study.

According to the integrated testing strategy for mutagenicity as described in the ECHA Guidance (Chapter R.7a, 2016, Table R.7.7-5) no further testing or information are required. The decision based on the negative results of the bacterial gene mutation tests (OECD TG 471) for Scopolamine and the read-across substance Scopolamine hydrobromide trihydrate and the negative result in a cytogenetic assay in experimental animals (in vivo) according to OECD TG 474 for the read-across substance Scopolamine hydrobromide trihydrate.

 

Justification for classification or non-classification

The available information on the read-across substance Scopolamine hydrobromide trihydrate indicates when tested in vitro that the substance does not induce mutations in bacterial cells but causes chromosomal aberrations in-vitro in the presence of metabolic activation. In an in vivo mouse micronucleus study with the read-across substance the clastogenic properties were not confirmed and the substance is concluded to be non clastogenic.

The available data on genotoxicity do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification of the registered substance.