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Diss Factsheets

Toxicological information

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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
publication
Title:
Mutagenicity Test Data of Existing Chemical Substances; based on the Toxicity Investigation System of the Industrial Safety and Health Law
Author:
JETOC (Japan Chemical Industry Ecology-Toxicology & Information Centre)
Year:
1996
Bibliographic source:
Chemical Substance Investigation Division, Industrial Safety and Health Department, Labour Standards Bureau, Ministry of Labor, Japan

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Method of Ames et al. (1975), Maron and Ames (1983) and Matsushima et al. (1980)
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
3,7-dinitroso-1,3,5,7-tetraazabicyclo[3.3.1]nonane
EC Number:
202-928-3
EC Name:
3,7-dinitroso-1,3,5,7-tetraazabicyclo[3.3.1]nonane
Cas Number:
101-25-7
Molecular formula:
C5H10N6O2
IUPAC Name:
3,7-dinitroso-1,3,5,7-tetraazabicyclo[3.3.1]nonane
Test material form:
solid
Details on test material:
- Chemical name: Dinitrosopentamethylenetetramine
- CAS: 101-25-7
- EC: 202-928-3
- MW: 186.17 g/mol
- pale yellow crystalline powder
Specific details on test material used for the study:
extra pure reagent (Tokyo Kasei Kogyo Co. Ltd), Lot AI01

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium, other: TA98, TA100, TA1535, TA1537, TA1538
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix (sodium phenobarbital and 5,6-benzoflavone induced)
Test concentrations with justification for top dose:
0, 50, 100, 200, 500, 1000, 2000, 5000 µg/plate
Vehicle / solvent:
DMSO
Controlsopen allclose all
Untreated negative controls:
other: Solvent control served as negative control
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S9
Untreated negative controls:
other: Solvent control served as negative control
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
sodium azide
other: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide
Remarks:
without S9
Details on test system and experimental conditions:
Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 were obtained from Dr. B.N. Ames (University of California, Berkeley, CA, USA), and Escherichia coli WP2uvrA were from Dr. M. Ishizawa (Kyushu University, Fukuoka, Japan). To a stationary culture of the tester strains, which had been checked for the genetic markers according to Ames et al. (1975) and Maron and Ames (1983), dimethyl sulfoxide (DMSO) was added to give a final concentration of 8.2% (v/v).
The cultures were stored in aliquots of 0.2mL at -80°C. The frozen permanents were thawed and inoculated to nutrient broth (OXOID, # 2) at 1 / 500-1 / 1000 inoculation size of thawed
solution. The cultures were incubated for 10-14 hours (tester strains grew up to early stationary phase) at 37°C in a shaking water bath before starting the tests.
Evaluation criteria:
Two-hold rule criteria was used for data evaluation (Ames et al., 1975). The chemicals are considered to be mutagenic when a dose-related increase in revertant colony count is observed and the number of revertant colonies per plate with the test substance is more than twice that of the negative control (solvent control) and when a reproducibility of test result is observed.
Mutagenic potency was calculated by following equation and maximum value of mutagenic potency was expressed as a specific acitivity on the data sheet:
mutagenic potency (induced revertants / mg test substance) = (number of induced revertants on the dose X - number of revertant on the solvent control) + mg of test chemical on the dose X.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Any other information on results incl. tables

Toxicity (*) was observed in all tester strains at 5000 µg/plate and in TA98 (+/-), TA100 (+), TA1535 (+/-), TA1537 (+/-) and TA1538 (+/-) at 2000 µg/plate.

Number of revertants per plate, without S9

µg/plate TA100 TA1535 E.coli WP2uvrA TA98 TA1537 TA1538
0 (DMSO) 179 36 24 26 16 13
50 202 23 22 25 15 13
100 179 16 18 14 16 8
200 165 19 25 17 24 8
500 263 16 30 22 313 11
1000 419 13 39 30 12 8
2000 0 * 0 * 32 0 * 0 * 0 *
5000 0 * 0 * 0 * 0 * 0 * 0 *
pos. control 1068 269 375 277 234 297

Number of revertants per plate, with S9

µg/plate TA100 TA1535 E.coli WP2uvrA TA98 TA1537 TA1538
0 (DMSO) 199 12 26 39 24 18
50 183 11 21 25 22 24
100 222 9 22 31 22 14
200 243 11 24 31 19 18
500 452 19 40 34 20 22
1000 825 15 51 61 12 13
2000 588 7 * 57 62 * 3 * 6 *
5000 0 * 0 * 18 * 0 * 0 * 0 *
pos. control 501 203 646 288 305 252

Applicant's summary and conclusion

Conclusions:
Under the conditions of this test, the test item was positive causing gene mutations by base pair changes in the genome of the tester strain S. typhimurium TA100 with and without metabolic activation.