Hyoscine

Endpoint summary

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

Reference

Reference 1

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

Justification for type of information:

The read-across justification is included as attachmant to Iuclid section 13.

Qualifier:

equivalent or similar to guideline

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.

Reference 2

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

Justification for type of information:

The read-across justification is included as attachmant to Iuclid section 13.

Qualifier:

equivalent or similar to guideline

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

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 1537

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 98

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 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	88^c	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 +-2.2^e	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:

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

Justification for type of information:

The read-across justification is included as attachmant to Iuclid section 13.

Qualifier:

equivalent or similar to guideline

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 or test system 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 weeks^a

	Dose (mg/kg)	Micronucleated NCEs/1000 Cells^b	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=0.714^c
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.