spirodiclofen (ISO);3-(2,4-dichlorophenyl)-2-oxo-1-oxaspiro[4.5]dec-3-en-4-yl 2,2-dimethylbutyrate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

not specified

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

Previously known as '92/69/EEC B.10'

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

1

Reference substance name:

3-(3,5-Dichlorophenyl)-2-oxo-1-oxaspiro[4.5]dec-3-en-4-yl 2,2-dimethylbutanoate

Cas Number:

148477-71-8

Molecular formula:

C21H24Cl2O4

IUPAC Name:

3-(3,5-Dichlorophenyl)-2-oxo-1-oxaspiro[4.5]dec-3-en-4-yl 2,2-dimethylbutanoate

Test material form:

not specified

Method

Target gene:

Not applicable

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Cytokinesis block (if used):

0.2 mL Colcemid-solution (40 ug/mL water) was added to each flask two hours prior to the end of the incubation period to arrest the cells in a metaphase-like stage of mitosis (c-metaphase).

Metabolic activation:

with and without

Metabolic activation system:

S9 mix was used for the simulation of the mammalian metabolism. The S9 fraction was isolated from the livers of Aroclor 1254 induced Wistar rats.

Test concentrations with justification for top dose:

5, 10, 20, 40, and 80 µg/mL without S9 mix.
10, 20, 40, 80 and 160 µg/mL with S9 mix.
Additional experiment: 0.75, 1.5, 3, 6 and 12 µg/ml without S9 mix.
Limited by cytotoxicity

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Spirodiclofen was tested for a clastogenic potential in an in vitro chromosome aberration test. Chinese Hamster V79 cells were exposed for 4 hours to spirodiclofen concentrations of 5, 10, 20, 40, and 80 µg/mL without S9 mix. With S9 mix 10, 20, 40, 80 and 160 µg/mL were used. In an additional performed experiment cells were exposed in the absence of S9 mix for 4 hours to concentrations of 0.75, 1.5, 3, 6 and 12 µg/ml of spirodiclofen. The positive controls were mitomycin C and cyclophosphamide.

Rationale for test conditions:

Test concentrations were based on the results of a preliminary assay.

Evaluation criteria:

An increased incidence of gaps of both types without concomitant increase of other aberration types was not considered as indication of a clastogenic effect. A test was considered positive if there was a relevant and statistically significant increase in the aberration rate. A test was considered negative if there was no such increase at any time interval. A test was considered equivocal if there was an increase which was statistically significant but not considered relevant,
or if an increase occurred, which was considered relevant, but which was not statistically significant. An assay was acceptable if there was a biologically relevant increase in chromosome aberrations induced by the positive controls and if the numbers of aberrations for the negative controls were in the expected range based on results from the laboratory and from published studies.

Statistics:

The statistical analysis was performed by pair-wise comparison of treated and positive control groups to the respective solvent control group. The mitotic index was statistically analyzed (provided that it was reduced compared to the respective negative control mean) using the one-sided chi-squared test. The numbers of metaphases with aberrations (including and excluding
gaps) and of metaphases with exchanges were compared (provided that these data superseded the respective negative control). The statistical analysis followed the recommendations outlined by Richardson et al. (1989). Fisher's exact test was used for the statistical evaluation. A difference was considered to be significant if the probability of error was below 5 %.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at >0.75 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at >20 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Without S9 mix, statistically significant values for the numbers of metaphases with aberrations were detected for 3 ug/mL and a culture time of 18 hours. These values were, however, within the range of historical negative controls. In addition, they were equal to the results of the untreated control for cultures harvested after a culture time of 30 hours, Therefore, these significances were not considered relevant. None of the other cultures neither after 18 nor after 30 hours culture time showed statistically significant or biologically relevant increased numbers of metaphases with aberrations. The treatment with the positive control mitomycin C resulted in a clear and statistically significant increase of metaphases with aberrations and demonstrated the sensitivity of the test system.

With S9 mix, no biologically relevant and statistically significant increases of metaphases with aberrations were detected after total culture times of 18 hours. For cultures treated with 80 ug/mL and terminated after a culture time of 30 hours significant values were observed for metaphases with aberrations. However, these values were within the range of historical negative controls and were, therefore, not regarded as a biologically relevant indication of a clastogenic effect. The positive control cyclophosphamide induced statistically significant and biologically relevant increases of metaphases with aberrations and demonstrated the sensitivity of the test
system and the activity of the used S9 mix.

Any other information on results incl. tables

Summary of results

Concentration (µg/mL)	Metaphases with aberrations (%)
	-S9		+S9
	18 hours	30 hours	18 hours	30 hours
0 (DMSO)	1.0	4.5	3.5	2.5
0 (untreated)	1.5	-	4.5	-
0.75	1.5	-	-	-
1.5	2.5	-	-	-
3	4.5*	4.0	-	-
20	-	-	1.5	-
40	-	-	3.5	-
80	-	-	2.0	-
MMC	44.0**	-	-	-
CPA	-	-	19.0*	7.0*

 

*significantly different to controls (p<0.05); **p<0.01)

Applicant's summary and conclusion

Conclusions:

No evidence of clastogenicity was seen under the conditions of this study.

Executive summary:

Spirodiclofen was tested for a clastogenic potential in an in vitro chromosome aberration test.  Chinese Hamster V79 cells were exposed for 4 hours to concentrations of 5, 10, 20, 40, and 80 μg/mL without S9 mix. With S9 mix 10, 20, 40, 80 and 160 μg/mL were used. In an additional performed experiment cells were exposed in the absence of S9 mix for 4 hours to concentrations of 0.75, 1.5, 3, 6 and 12 μg/mL of spirodiclofen. The positive controls were mitomycin C (-S9) and cyclophosphamide (+S9). Cytotoxic effects were seen at 0.75 μg/mL and above (-S9) and at 20 μg/mL and above (+S9). None of the cultures treated with spirodiclofen in the absence and in the presence of S9 showed biologically relevant increased numbers of aberrant metaphases. The positive controls demonstrated a good sensitivity of this test system. In conclusion, spirodiclofen was not considered to be clastogenic for mammalian cells with and without metabolic activation in vitro.