3-[(aminoiminomethyl)thio]propanesulphonic acid

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1994-06-09 - 1994-06-27 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

study combines both OECD 471 and 472 of 1983, covering hence all strains as required in the OECD 471 of 1997

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

OECD Guidelines for Testing of Chemicals No. 471 Genetic Toxicology Salmonella typhimurium, Reverse Mutation Assay, 26 May 1983

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Version / remarks:

OECD Guidelines for Testing of Chemicals No. 472: Genetic Toxicology: Escherichia coli, Reverse Mutation Assay, 26 May 1983,

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EEC Methods for Determination of Toxicity, Annex to Directive 92/69/EEC, (OJ No. L383A, 29.12.92), Part B, Method B.14. Other effects - Mutagenicity: Salmonella typhimurium - Reverse Mutation Assay

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: EEC Methods for Determination of Toxicity, Annex to Directive 92/69/EEC, (OJ No. L383A, 29.12.92), Part B, Method B.13. Other effects - Mutagenicity: Escherichia coli - Reverse Mutation Assay

Deviations:

no

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: US Environmental Protection Agency, Method: HG-Gene Muta - S. typhimurium: The Salmonella typhimurium reverse mutation assay, 1984

Deviations:

no

Test guideline 6

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

Japanese, Ministry of Agricultural, Forestry and Fisheries: Notification of Director General, Agricultural Production Bureau, NohSan 4200, 28 January 1985
Japanese, Ministry of Health and Welfare, Notification Yakushin 1 No. 24, 11
September 1989 Guidelines for Toxicity Studies of Drugs, 4 I, Bacterial Reverse Mutation Test
Japariese, Ministry of International Trade & Industry, 61 Kilcyolcu No. 1014, 5 December 1986 and 62 Kilcyoku No. 303, 31 March 1987
Japanese, Ministry of Labour, Guidebook of Mutagenicity Tests, published 16 June 1987.

Deviations:

no

Test guideline 7

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

study combines both OECD 471 and 472 of 1983, covering hence all strains as required in the OECD 471 of 1997

Deviations:

not applicable

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3-[(aminoiminomethyl)thio]propanesulphonic acid

EC Number:

244-520-8

EC Name:

3-[(aminoiminomethyl)thio]propanesulphonic acid

Cas Number:

21668-81-5

Molecular formula:

C4H10N2O3S2

IUPAC Name:

3-(carbamimidoylsulfanyl)propane-1-sulfonic acid

Test material form:

solid: particulate/powder

Details on test material:

Storage: Room temperature under desiccating conditions

Method

Target gene:

his- resp. trp-

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: The strains were obtained from Professor B.N.Ames, University of California, California, U.S.A..
The following strains of S. typhimurium were used in the test:
S. typhimurium TA 1535 hisG46 rfa uvrB
S. typhimurium TA 1537 hisC3076 rfa uvrB
S. typhimurium TA 1538 hisD3052 rfa uvrB
S. typhimurium TA 98 hisD3052 rfa uvrB pl(M101
S. typhimurium TA 100 hisG46 rfa uvrB pl(M101
The strain of E. coli used was:
E. coli WP2 uvrA trp

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: The strains were obtained from Professor B.N.Ames, University of California, California, U.S.A..
S. typhimurium TA 1538 hisD3052 rfa uvrB

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced mal rat (Sprague-Dawley derived) liver S9

Test concentrations with justification for top dose:

Top dose was 5000 µg/plate due to results of cytotoxicity test.
Doses were 5000, 2500, 1250, 625, 312.5, and 0 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Prior to commencing testing the solubility of the test substance was assessed. At 50 mg/ml UPS was soluble in water. Therefore water chosen was the chosen solvent for the subsequent tests. This solvent was also recommended by the Study Sponsor.

Controls

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding (if applicable): 0.1 ml of approx. 2 x 10exp9 cells per ml per plate

DURATION
- Preincubation period: none
- Exposure duration: 3 days

SELECTION AGENT (mutation assays): histidine/tryptophan deficient agar

NUMBER OF REPLICATIONS: Three petri dishes were used for each dose level, two experiments were performed

DETERMINATION OF CYTOTOXICITY
- Method: Any toxic effects of the test substance can be detected by a substantial reduction in revertant colony counts or by the absence of a complete background bacterial lawn.

Evaluation criteria:

The mean number of revertant colonies for all treatment groups is compared with those obtained for solvent control groups. The mutagenic activity of a test substance is assessed by applying the following criteria:
(a) If treatment with a test substance produces an increase in revertant colony numbers of at least twice the concurrent solvent controls, with some evidence of a positive dose-relationship, in two separate experiments, with any bacterial strain either in the presence or absence of S-9 mix, it is considered to show evidence of mutagenic activity in this test system. No statistical analysis is performed.
(b) If treatment with a test substance does not produce reproducible increases of at least 1.5 times the concurrent solvent controls, at any dose level with any bacterial strain, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.
(c) If the results obtained fail to satisfy the criteria for a clear "positive" or "negative" response given in paragraphs (a) and (b), the following approach is taken in order to resolve the issue of the substance's mutagenic activity in this test system.
(d) Repeat tests may be performed using modifications of the experimental method. These modifications include (but are not restricted to), the use of a narrower dose range than that already tested; the use of different levels of liver homogenate S-9 fraction in the S-9 mix. Should an increase in revertant colony numbers be observed which satisfies paragraph (a) the substance is considered to show evidence of mutagenic activity in this test system. No statistical analysis is performed.
(e) If no clear "positive" response can be obtained the test data may be subjected to analysis to determine the statistical significance of any observed increases in revertant colony numbers. The statistical procedures used will be those described by Mahon et al. (1989).

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: not exceeded
- Precipitation: none stated

RANGE-FINDING/SCREENING STUDIES:
UPS was not toxic towards the tester strains. Therefore 5000 µg/plate was chosen as the top dose level in the mutation tests.

Applicant's summary and conclusion

Conclusions:

Testing was performed via a GLP OECD 471 and 472 guideline study on the registered substance itself. The study combines both OECD 471 and 472 of 1983, covering hence all strains as required in the OECD 471 of 1997. The validity criteria are met, making the results sufficiently reliable to assess the mutagenic potential of the test item towards bacteria. No substantial increases in revertant colony numbers of any of the tester strains were observed following treatment with UPS at any dose level, in the presence or absence of S-9 mix, in either mutation test. It is concluded that, when tested in water, UPS shows no evidence of mutagenic activity in this bacterial system.

Executive summary:

In this in vitro assessment of the mutagenic potential of UPS, performed via a GLP OECD 471 and 472 guideline study, histidine dependent auxotrophic mutants of Salmonella typhimurium (strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100) and a tryptophan dependent mutant of Escherichia coli (WP2 uvrA) were exposed to the test substance, diluted in water which was also used as a negative control.

Two independent mutation tests were performed, in the presence and absence of liver preparations from Aroclor 1254-induced rats.

In the preliminary dose range finding study with dose levels of up to 5000 µg/plate no toxicity was observed. A top dose level of 5000 µg/plate was chosen for the subsequent mutation study. Other dose levels used in the mutation assays were: 2500, 1250, 625, 312.5 µg/plate.

No evidence of mutagenic activity was seen at any dose level of UPS in either mutation test. The concurrent positive control compounds demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations.

It is concluded that, when tested in water, UPS was not mutagenic in this bacterial system.