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Genetic toxicity: in vitro

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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 guidelineopen allclose all
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
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
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
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
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
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
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

Constituent 1
Chemical structure
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 / strainopen allclose all
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 / 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 resultsopen allclose all
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
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
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
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
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
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.