Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Aug 30, 1995 to October 27, 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1955

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
gas under pressure: refrigerated liquefied gas
Details on test material:
Identification: HEOX-CN
Chemical Name: 1-0xa-4-azaspiro[4.5]decane-4-ethanol
Cas-number : 2359-11-7
Description Clear liquid at 30°C
Batch: 0029504011005
Purity: (purity: 98.9+/- 0.4 % m/m; cyclohexanone: 0.50 +/- 0.15% m/m;
diethanolamine: 0.15 +/- 0.10 % m/m)
Test substance storage: In refrigerator, dry and in the dark
Stability under storage
conditions: Stable
Expiry date: December 01, 1995
Density: 1050 kg/m3 (40°C)
Preparation The test substance was dosed undiluted as delivered by the sponsor
Stability in vehicle Dimethylsulphoxide; not indicated

Method

Target gene:
Histidine operon for Salmonella
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction was obtained from livers of rats treated with Aroclor 1254 (500 mg/kg body weight) in corn oil
Test concentrations with justification for top dose:
Expt 1: 3, 10, 33, 100, 333, 1000, 3330, and 5000 ug/plate.
Expt 2: 100, 333, 1000, 3330, and 5000 ug/plate.
Vehicle / solvent:
dimethylsulphoxide
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
test item in dimethylsulphoxide
Positive controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
described under test system conditions
Positive control substance:
9-aminoacridine
Remarks:
described under test system conditions
Positive control substance:
methylmethanesulfonate
Remarks:
described under test system conditions
Positive control substance:
other: 2-aminoanthracene
Remarks:
described under test system conditions
Positive control substance:
other: daunomycine
Remarks:
described under test system conditions
Details on test system and experimental conditions:
Positive controls:
Without metabolic activation
Strain Chemical Solvent
TA1535 sodium azide (SA) (Fluka) 1 ug Saline
TA1537 9-aminoacridine (9AC) 60 ug Saline
(Janssen Chimical)
TA98 daunomycine (DM) (Sigma) 4 ug Saline
TA100 methylmethanesulfonate (MMS) 650 ug DMSO
(Merck)

With metabolic activation
Strain Chemical Sol vent
TA1535 and
TA1537 2-aminoanthracene (2AA) 5 ug DMSO
(Sigma)
TA 98 and
TA100 2-aminoanthracene (2AA) 0.5ug DMSO
(Sigma)

Solvents for reference substances:
Saline = physiological saline (Medital Pharma Ned. B. V.)
DMSO = dimethylsulphoxide of spectroscopic quality (Merck)
Evaluation criteria:
ACCEPTABILITY OF ASSAY
A Sal monel l a typhimurium reverse mutation assay is considered acceptabl e if it meets the fol l owing criteria:
a) The negative control data (number of spontaneous revertants per plate) should reasonably be within the laboratory background historical range
for each tester strain.
b) The positive control chemicals should produce responses in all tester strains which also are reasonabl y within the laboratory historical range documented for each positive control substance. Furthermore, the mean plate count should be at least 2 times the concurrent vehicle control group mean
c) The sel ected dose range shoul d include a cl earl y toxic concentration as demonstrated by the prel iminary range-finding test with strain
TAI00 or shoul d extend to 5 mg/pl ate.

DATA EVALUATION AND STATISTICAL PROCEDURES
No formal hypothesis testing was done.
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the sol vent control val ue, with or
without metabol ic activation.
b) The negative response shoul d be reproducibl e in at l east one independentl y repeated experiment.
A test substance is considered positive (mutagenic) in the test if:
a) It induces at l east a 2-fol d, dose rel ated increase in the number of revertants with respect to the number induced by the solvent control in
any of the tester strains, either with or without metabol ic activation. However, any mean pl ate count of l ess than 20 is considered to be not
significant. If in the first test the test substance shows only a positive response at one or two concentrations, the assay was repeated
with doses just below and exceeding those showing positive effects in the first test.
b) Positive response should be reproducible in at l east one independentl y repeated expt. The preceding criteria were not absolute and other modifying factors might enter into the final decision.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS
SELECTION OF DOSE LEVELS: HEOX-CN was tested in strain TA100 with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 􀄷g/plate in the absence and presence of S9-mix. This prel iminary test is reported as a part of the first experiment of the mutation test (Tabl e 1) . The individual data are presented in Appendix 2 of the report.

Precipitate
HEOX-CN did not precipitate in the top agar. Precipitation of HEOX-CN on the pl ates was not observed at the start or at the end of the incubation period in tester strain TA100.

Toxicity
No reduction of the bacterial background l awn and no decrease in the number of revertants was observed.

Based on these data, HEOX-CN was tested in the mutation assay up to and including a concentration of 5000 􀄷g/pl ate in the absence and presence of S9-mix.

MUTATION ASSAY
Tables 1 and 2 show the results of the Sal monella typhimurium reverse mutation assay with HEOX-CN. The individual data are presented in Appendix 2 of the report.

Precipitate
HEOX-CN did not precipitate in the top agar. Precipitation of HEOX-CN on the pl ates was not observed at the start or at
the end of the incubation period in al l tester strains.

Toxicity of the test substance
The bacterial background l awn was not reduced at al l concentrations tested and no decrease in the number of revertants was observed.

Number of revertants
All bacterial strains showed negative responses over the entire dose range, i. e. no dose-related, two-fold, increase in the number of revertants in two
independentl y repeated experiments. The negative and strain-specific positive control values were within our laboratory background historical ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

REFERENCES

1) Ames, B. N. , McCann, J. and Yamasaki, E., 1975, Methods for detecting carcinogens and mutagens with the Salmonella / mammalian microsome mutagenicity test, Mutation Res. , 31, 347 -364.

2) Maron, D. M. and Ames, B. N. , 1983, Revised methods for the Sal monel la mutagenicity test, Mutation Res. , 113, 173-215. Erratum, 1983, Mutation Res. , 113, 533.

3) Vogel , H. J. and Bonner, D. M. , 1956, Acetyl ornithinase of Escherichia coli: partial purification and some properties. J. Biol . Chem. , 218, 97-106.

Applicant's summary and conclusion

Conclusions:
Based on the resul ts of this study it is concl uded that HEOX-CN is not mutagenic in the Sal monella typhimurium reverse mutation assay.
Executive summary:

The study procedures described in this report were based on the OECD Guideline no. 471: 'Genetic Toxicol ogy: Salmonella typhimurium Reverse Mutation Assay', (adopted May 26, 1983) and the EEC, Directive 92/69/EEC. Annex V of the EEC Directive 67/548/EEC, Part B: Methods for the Determination of Toxicity; B.14: 'Other Effects- Mutagenicity: Salmonel l a typhimurium - Reverse Mutation Assay'. December, 1992. The objective was to evaluate the test substance for its ability to induce reverse mutations in a gene of histidine- requiring Salmonella typhimurium bacterial strains to produce histidine - independent strains of these micro - organisms. JThis test system has been shown to be a rapid and adequate indicator for the mutagenic activity of a wide range of chemical compounds. The assay was conducted in the absence and in the presence of a metabolic system (S9 -mix) . Test strains used in this study were: TA98, TAI00, TA1535 and TA1537. The strains TA98 and TA1537 are capable of detecting frameshift mutagens; and strains TAI00 and TA1535 are capable of detecting base-pair substitution mutagens.

HEOX-CN (CAS# 2359 -11 -7) was tested in the Salmonella typhimurium reverse mutation assay with four histidine - requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) in two independent experiments. HEOX-CN was tested up to and including concentrations of 5000 ug/plate in the absence and presence of S9 -mix. HEOX-CN did not induce a dose-related increase in the number of revertant colonies in the absence or in the presence of S9 - mix, in two independentl y repeated experiments.

Based on the results of this study it is concluded that HEOX-CN is not mutagenic in the Salmonell a typhimurium reverse mutation assay.