2-butyl-1,3-diazaspiro[4.4]non-1-en-4-one hydrochloride

Genetic toxicity: in vitro

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:

8 February to 28 March, 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted according to OECD method and in accordance with GLP. Study material is well characterized.

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)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Method

Target gene:

five histidine-requiring strains

Species / strain

Species / strain / cell type:

bacteria, other: Salmonella typhimurium: TA1535, TA1537, TA98, TA100, TA102

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix(2.5%) from Aclor 1254-induced rat liver

Test concentrations with justification for top dose:

Preliminary toxicity (Range finding) on TA 98 experiment carried out at concentrations of 0, 100, 250, 500, 1000, 2500 and 5000 ug/plate ( preincubation and plate incorporation) . For genotoxicity experiment concentrations (with & without metabolic activation) used: 0, 250, 500, 1000, 2500 and 5000 ug/plate.

Vehicle / solvent:

Solvent: Distilled water ( 100 ul/plate)

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

vehicle controls used in parallel with the test material

Controls 2

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Migrated to IUCLID6: mitomycin c: 0.25 ug/plate (TA102) without s-9.

Controls 3

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 1 ug/plate(TA100), 2.5 ug/plate (TA 98,TA1535&TA1537)

Controls 4

Positive controls:

yes

Positive control substance:

other: danthron 10 ug/plate (TA102) with s-9

Controls 5

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Migrated to IUCLID6: 1 ug/plate (TA100,TA1535) without s-9.

Controls 6

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Migrated to IUCLID6: 9-aminoacridine:100 ug/plate (TA1537) without s-9.

Controls 7

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Migrated to IUCLID6: 2-nitrofluorene:2.5 ug/plate (TA98) without s-9.

Details on test system and experimental conditions:

A toxicity range finding experiment was conducted with strain TA98 six concentrations plus negative (vehicle) controls in absence and in the presence of metabolic activation S-9 with both methods used (preincubation and plate incorporation method). No evidence of toxicity was observed following this treatment. This test was acceptable and no evidence of toxicity was observed following this treatment. For main test five concentrations were assayed in triplicate against each tester strain with and without metabolic activation following preincubation method. No evidence of toxicity was observed following this treatment. Negative and positive control treatments were included. The mean numbers of revertant colonies on negative control plates were all acceptable.

Evaluation criteria:

Evaluation criteria: test article would be considered mutagenic if:
the increase in the number of revertants is concentration-related;
at one concnetration tested ( at least), the number of revertant colonies is equal to or greater than twice the number of spontaneous revertants with TA 98, TA 100 and TA 102 and three times that observed with TA1535 and TA1537;
the positive responses described above were reproducible in an independent assay.

Statistics:

Mean and standard deviation of the plate counts for each treatment were determined.

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

other: preliminary test

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(> 5000 µg/plate)

Test results 2

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

other: preliminary test

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(> 5000 µg/plate)

Test results 3

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:

cytotoxicity

Remarks:

(> 5000 µg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(> 5000 µg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Observations:
All positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9 mix were validated.

The test substance caused no visible reduction in the growth of the bacterial lawn at any dose level. The test substance was, therefore, tested up to the maximum recommended dose of 5000 ug/plate. No test substance precipitate was observed on the plates at any doses tested in either the presence or absence of S9 mix.

Remarks on result:

other: other: preliminary test

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative without metabolic activation
negative without metabolic activation

No dose-related and reproducible increases in revertant colony frequency were observed in any tester strains at any concentration, both with and without S9. The test substance was not genotoxic in the Ames test.