1,3-propanesultone

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A high potential for genetic toxicity of the test item was indicated in in vitro test systems (e.g. Ames test, chromosomal aberration test, micronucleus test).

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:

1975

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

not applicable

GLP compliance:

no

Type of assay:

other: in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

mammalian cell line, other: Chinese hamster lung fibroblast cell line (CHL)

Details on mammalian cell type (if applicable):

- originally established from the lung of a young adult by Dr. T. Utakoji, Cancer Institute, Tokyo
- karyotype consists of 25 chromosomes
- maintained by 5-day passages
- grown in a monolayer in petri dishes with Eagle's MEM (GIBCO F-11) supplemented with 10 % calf serum
- doubling time was estimated as 18.2 h at their exponential growth at 37 °C in a 5 % CO2 atmosphere

Metabolic activation:

without

Test concentrations with justification for top dose:

0.0313 , 0.0625 or 0.125 mg/mL

Vehicle / solvent:

- physiological saline

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

solvent: physiological saline

True negative controls:

no

Positive controls:

yes

Positive control substance:

not specified

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 24 and 48 h
- Fixation time (start of exposure up harvest of cells): 24 and 48 h

SPINDLE INHIBITOR (cytogenetic assays): colcemid (0.2 µg/mL; added to the medium 2 h before harvest)
STAIN (for cytogenetic assays): 1% Giemsa's buffered solution (pH 6.8)

NUMBER OF REPLICATIONS: no data

NUMBER OF CELLS EVALUATED: the number of cells with chromosomal aberrations was recorded on 100 well-spread metaphases

DETERMINATION OF CYTOTOXICITY
- only examinded in a pre-test (growth inhibition test)
- the dose inducing a 50% growth inhibition was taken as the highest dose in the main test

Growth inhibition tests carried out before the chromosome tests were started:
The 50% growth inhibition dose was estimated as follows:
Several different doses of each agent were separately added to the 3-day-old cultures (about 6 x 10E3 cells/3-cm dishes). The doses were prepared by a factor of 2 from the maximal dose, estimated from the data on LD50, which appeared in references for the test item. The cells in a monolayer were washed, fixed with 10% formalin solution, and then stained with 0.1% crystal violet solution for 3 min. After washing and drying each dish was placed under a photodensitometer to measure the color absorption values from which relative cell densities on the dishes were easily calculated. The color absorption values obtained reflected well the actual number of cells that survived at the bottom of each petri dish.


OTHER EXAMINATIONS:
- Determination of polyploidy: yes

OTHER:
- types of aberration were classified into 5 groups: chromatid gaps, chromatid breaks, chrorrmatid or chromosomal translocation, ring formation and fragmentation or pulverization

Evaluation criteria:

CHL cells commonly have less than 3.0% cells with chromosomal aberrations. Therefore, the final judgement given to all experimental groups was as follows: Negative if less than 4.9% of the aberration was detected – even when doses of the agent were elevated to sub-lethal amounts, where almost no mitosis was observed; suspicious if between 5.0 and 9.9%, and positive if between 10.0 and 19.9% (+), 20.0 and 49.9% (++) and more than 50.0% (+++). When no reasonable dose response was obtained, experiments with different doses were carried out to confirm its reproducibility.

Statistics:

no data

Key result

Species / strain:

mammalian cell line, other: Chinese hamster fibroblast cell line (CHL)

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

94% cells with abberations

Cytotoxicity / choice of top concentrations:

other: cytotoxicity can be assumed as the dose where 50% growth inhibition occured in the pre-test was used as the highest dose

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not examined

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- no data

COMPARISON WITH HISTORICAL CONTROL DATA:

- CHL cells commonly have less than 3.0% cells with chromosomal aberrations. This value was taken for judgement.

A D20 value (the dose (mg/mL) at which chromosomal aberrations were detected in 20% of metaphases) of 0.0451 mg/mL was obtained.

Results chromosome test (only 24 values are presented):

Dose [mg/mL]	No of Metaphases	Polyploid cells [%]		Cells with structural chromosome aberrations [%], 24 h
		24 h	48 h	G	B	T	R	F		Total
None	100	1	-	1	0	0	0	0		1
Solv		-	-	-	-	-	-	-		-
0.0313	100	0	1	1	3	2	0	0		6
0.0625	100	1	0	2	7	16	0	0		22
0.125	100	1	T	0	46	91	0	0		94

G: chromatid gaps

B: chromatid or chromosomal breaks

T: translocation

R: ringformation

F: fragmentation

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not applicable

Principles of method if other than guideline:

- test was conducted only in absence of S9 mix at one test concentration

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

not applicable

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

S. typhimurium TA 1538

Species / strain / cell type:

other: S. typhimurium TA 1536

Metabolic activation:

without

Test concentrations with justification for top dose:

5 µg per plate

Vehicle / solvent:

no data

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

1 µg

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- in agar (plate incorporation)


NUMBER OF REPLICATIONS:
- no data


DETERMINATION OF CYTOTOXICITY
- Method: colonies were counted

Evaluation criteria:

Mutagens were defined as chemicals that induced a reproducible dose-related increase in the number of histidine-independent revertants.

Statistics:

not applicable

Species / strain:

S. typhimurium, other: TA 1535, TA 100

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA 1536, TA 1537, TA 98 and TA 1538

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Revertants/ plate after subtacting background:
TA 1535: 170 (vehicle control: 25 - 55)
TA 100: 290 (vehicle control: 100 - 180)

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not applicable

Principles of method if other than guideline:

- the test item was only tested in the absence of S9 mix at unspecified concentrations

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

not applicable

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Species / strain / cell type:

S. typhimurium TA 97

Metabolic activation:

without

Test concentrations with justification for top dose:

no data

Vehicle / solvent:

no data

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

not specified

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: methylnitronitrosoguanidine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

Species / strain:

S. typhimurium, other: TA 1535, 1537, 98, 100 and 102

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

the frequency of induced mutants was more than 2-5 fold the spontaneous background

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 97

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

the frequency of induced mutants was more than 2-5 fold the spontaneous background

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The frequency of induced mutants was more than 2-5 fold the spontaneous background.

Reference 4

Endpoint:

genetic toxicity in vitro, other

Remarks:

Summary of various types of genotoxicity tests

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: only result is given without any further detail

GLP compliance:

no

Summary: Genotoxicity studies and related tests with 1,3-propane sultone

 

Test system	Dose or concentration	Test result	Observations	Ref.
in vivo/in vitro transformation assay (hamster embryo cells exposed transplacentally)	1.0 - 3.0 mg/ 100 g i.p.	+	neoplastic transformation in cultured cells obtained from embryos after transplacental exposure	DiPaolo et al., 1973
inhibition of in vivo^3H-thymidine incorporation in kidneys of suckling mice	0.1 mL/mouse (c 15-30% of LD50), i.g.	+	marked inhibition of DNA synthesis in kidney cells	Amlacher et al., 1983
cell transformation test in hamster embryo fibroblasts	0.1, 1.0, 10 mg/L	+	cell transformation at 1 mg/l; results negative with lower and higher concentrations	Pienta et al., 1977
cell transformation test in C3H/10T½CL8 cells	25 - 100 µg/mL	+	small number of transformed foci with highly toxic concentrations	Oshiro et al., 1981
cell transformation test in C3H/10T½CL8 cells	20 - 60 µg/mL	+	marked dose-dependent transformation response	Nesnow et al., 1982
reversion assay in S. typhimurium TA1535, TA1538	5 - 25 µg/plate	+	dose-dependent increase in reversions in TA1535; effect effect reduced slightly by S9 mix; no effect in TA1538	Rosenkranz et al., 1971
6 tests in combination:	various (appropriate for the test system)		all tests yielded positive results apart from mouse skin sebaceous gland suppression test	Purchase et al., 1978
Ames test in S. typhimurium (4 strains); mammalian cell transformation test (BHK21/cl13 Syrian hamster kidney cells)		+ +
degranulation of rat liver rough endoplasmic reticulum;		+
tetrazolium reduction test;		+
mouse skin sebaceous gland suppression test; subcutaneous implantation test		- +
reversion test in Escherichia coli 343	1 mg/mL	+	reversion of mutants unable to utilize galactose	Mohn, 1971
reversion test in E. colli WP2	n.s.	+	reversion of tryptophan-auxotrophic mutants	Dean, 1972
assay of differential growth of repair-defective mutants and wild type Proteus mirabilis	610 µg/plate	+	slightly more inhibition of growth of repair-defective mutants than of wild type	Adler et al., 1976
reversion test in Serratia marcescens α13 and α21	25 mg/mL (in DMSO)	+++	reversion of histidine (α13) and leucine (α21) auxotrophic mutants	Dean, 1972
forward and back mutation assays in bacteriophage T4	1.6 mg/mL	+	various types of point mutation, mostly base pair transitions; very potent mutagen	Corbet et al., 1970
mitotic recombination assay in Saccharomyces cerevisiae D3	0.1% (w/v)	+	increased recombination frequency	Simmon, 1979
reversion test Schizosaccharomyces pombe	2 - 15 mM	+	dose-dependent increase in reversions in auxotrophic mutants	Heslot, 1962

 

 

References:

 

DiPaolo, J.A., R.L. Nelson,P.J. Donovan, Ch. H. Evans: Arch. Path. 95, 380 (1973)

Amlacher, E., Ch Rudolph: Arch. Geschwulstforsch. 51, 603 (1981)

Pienta, R.J., J.A. Poiley, W.B. Lebherz: Int. J. Cancer 19, 642 (1977)

Oshiro, Y., P.Sl Balwierz, S. V. Molinary: Toxicol. Lett. 9, 301 (1981)

Nesnow, S., H. Garland, G. Curtis: Carcinogenesis 3, 377 (1982)

Rosenkranz, R.S., L.A. Poirier: J. nat. Cancer Inst. 62, 873 (1971)

Purchase, I. F. H., E. Longstaff, J., J.Ashby, J.A. Styles, D. Anderson, P.A. Lefevre, F.R. Westwood: Brit. J. Cancer 37, 873 (1978)

Mohn, G.: Arch. Toxikol. 28, 93 (1971)

Adler, B., R. Brauch, J. Schöneich, H. Böhme: Biol. Zbl. 95, 463 (1976)

Corbett, Zh H., C. Heidelberger, F. D. Dove: Molec. Pharmacol. 6, 667 (1970)

Simmon, V. F.: J. nat.. cander Inst. 62, 893 (1979)

Heslot, H.: Abhandl. deut. Akad. Wiss. Berlin Kl. Med., 193, (1962)

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

A high potential for genetic toxicity of the test item was indicated in in vivo test systems (e.g. Micronucleus test, DNA damage).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

not applicable

GLP compliance:

not specified

Type of assay:

other: micronucleus assay

Species:

mouse

Strain:

CD-1

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Diet (ad libitum): commercial pellets
- Water (ad libitum): yes

ENVIRONMENTAL CONDITIONS
- no data

Route of administration:

intraperitoneal

Vehicle:

- saline

Duration of treatment / exposure:

- animals were treated with the test substance twice (24 h intervall) and peripheral blood was collected up to 72 h after last treatment

Frequency of treatment:

- twice (24 h intervall)

Post exposure period:

- 24, 48, 72 h

No. of animals per sex per dose:

- 5 male mice per dose

Control animals:

yes

Positive control(s):

- Mitomycin C
- Route of administration: ip
- Doses / concentrations: 0.5 mg/kg (single dose)

Tissues and cell types examined:

- peripheral blood reticulocytes were used for analyses

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
- The highest dose was fixed by the preliminary dose-finding test (based on mortality)

METHOD OF ANALYSIS:
- Micronucleated reticulocyte frequencies were based on the observation of at least 1000 polychromatic reticulocytes

Statistics:

When the control data were acceptable, the increase in micronucleus frequency against the concurrent negative control data were evaluated using a conditional binomial test and the dose response relationship using the Cochran-Armitage trend test were evaluated. When these were both significant, the data was declared positive. If neither step showed significance, the data was judged negative. All other cases were called inconclusive.

Key result

Sex:

male

Genotoxicity:

positive

Toxicity:

yes

Remarks:

can be assumed, since the highest dose tested was fixed by a preliminary dose-finding test

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Summary of the micronucleus assay results:

% micronucleated reticulocytes
dose	0 h	24 h		48 h		72 h
mg/kg bw	mean SD	mean SD	p	mean SD	p	mean SD	p
9	0.08± 0.04	0.17± 0.10	0.151	0.10± 0.13	0.500	0.08± 0.10	0.623
18	0.10± 0.11	0.23± 0.10	0.058	0.10± 0.11	0.613	0.07± 0.05	0.828
36	0.12± 0.08	0.87± 0.53	< 0.001	0.63± 0.16	< 0.001	0.28± 0.16	0.032
72	0.10± 0.09	1.92± 0.99	< 0.001	1.68± 0.50	< 0.001	0.27± 0.14	0.026

p: value of the pairwise comparison

Reference 2

Endpoint:

genetic toxicity in vivo, other

Remarks:

Summary of various types of genotoxicity tests

Type of information:

other: Review

Adequacy of study:

supporting study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Only secondary source.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Mode of Action Analysis / Human Relevance Framework

The considerable mutagenic potency of the alkylating agent of 1,3-propanesultone – demonstrated in a large number of test systems in vitro and in vivo – and its ability to induce various systemic tumours (see also chapter Carcinogenicity) shows that it is an unequivocally mutagenic substance with potent germ cell mutagen properties. No metabolic activation is needed to evolute its mutagenic properties.

Additional information

Ames-test:

In a reverse gene mutation assay the bacteria strains TA 1535, TA 1536, TA 1537, TA 1538, TA 98 and TA 100 of S. typhimurium were exposed to 1,3-propanesultone at a concentration of 5 µg/plate in the absence of mammalian metabolic activation applying the standard plate incorporation method (Simmon, 1979). The test item was found to be mutagenic in tester strains TA 1535 and TA 100 (170 and 290 revertants per plate after subtracting the background, respectively).

In another reverse gene mutation assay the bacteria strains TA 1535, TA 97, TA 1537, TA 102, TA 98 and TA 100 of S. typhimurium were exposed to 1,3-propanesultone in the absence of mammalian metabolic activation applying the standard plate incorporation method (Khudoley, 1987). The test item was found to be mutagenic in all tester strains (the frequency of induced mutants was more than 2 - 5 fold the spontaneous background).

Chromosomal aberration in vitro:

An in vitro assay for chromosomal damage was conducted in CHL cells to identify the potential 1,3-propanesultone to induce chromosomal aberrations (Ishidate, 1977). CHL cells were exposed to the test item at concentrations of 0, 0.0313, 0.0625 or 0.125 mg/mL without metabolic activation. A positive result for the test item was obtained as in 94 % of the cells analysed chromosomal aberrations were detected.

Micronucleus test in vitro and in vivo:

The potential of 1,3-propanesultone to induce micronuclei in vitro was examined in primary cultured astrozytes derived from 4 - 5 day old rats in the absence of metabolic activation (Ooida et al., 2000). After an exposure time of 24 hours the cells were fixed, stained and examined for mironuclei (MN). The frequencies of MN cells obtained in the dose-response study increased significantly at 0.08 (p < 0.05), 0.16 (p < 0.01) and 0.31 mM (p < 0.01). A dose-response relationship of the frequency of MN cells was observed up to 0.31 mM 1,3-propanesultone. Frequencies of MN cells decreased at 0.63 mM because of toxicity. The 50% growth inhibition ratio of 1,3-propanesultone was observed at 0.56 mM.

An in vivo micronucleus assay in mice was performed with 1,3-propanesultone given twice intraperitoneally (24-hour interval) at doses of 0, 9, 18, 36 or 72 mg/kg bw (Morita et al., 1997). 24, 48 and 72 hours after last application peripheral blood reticulocytes were sampled and analysed for micronulei. 1,3-propanesultone induced statistically significant increases (p<0.05) of micronucleated cells at all dose time points at doses of 36 and 72 mg/kg bw.

Other tests on genotoxicity:

Numerous other tests on genetic toxicity in vitro and in vivo (MAK, 1985; Raschig, 2009) indicate also a high mutagenic potential of 1,3-propanesultone.

Justification for classification or non-classification

The available data show that 1,3-propanesultone is mutagenic in various in vitro and in vivo test systems. Thus, 1,3-propanesultone is subject for classification and labelling according to Regulation 1272/2008/EC (Cat. 2, H341) regarding mutagenicity.