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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A GLP- and guideline (OECD 471) compliant Ames test is available for spirodiclofen.

A GLP- and guideline (OECD 473) compliant chromosomal aberration assay in V79 cells is available for spirodiclofen.

A GLP- and guideline (OECD 476) compliant mammalian cell mutation (HPRT) assay in V79 cells is available for spirodiclofen.

All three assays report negative results.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

not specified

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Deviations:

no

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

Target gene:

Not applicable

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

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 %.

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

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.

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)

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.

Reference 2

Endpoint:

in vitro gene mutation 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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: 88/302/EEC

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Cytokinesis block (if used):

Not applicable

Metabolic activation:

with and without

Metabolic activation system:

Male Sprague Dawley rats, induced by Aroclor 1254, served as the source of the S9 fraction. The rat liver S9 fractions were commercially purchased.

Test concentrations with justification for top dose:

Up to 80 µg/mL with S9 mix.
Up to 20 µg/mL without S9 mix.
The concentration range of spirodiclofen for the mutagenicity study was chosen according to the results of the cytotoxicity test with spirodiclofen.

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

other: Dimethylbenzanthracene

Remarks:

Ethylmethanesulfonate (EMS) - without S9 mix.
Dimethylbenzanthracene (DMBA) - with S9 mix.

Details on test system and experimental conditions:

Spirodiclofen was tested for mutagenic effects at the hypoxanthine-guanine phosphoribosyl transferase locus (forward mutation assay) in V79 cell cultures in vitro with concentrations of up to 80 µg/mL with and of up to 20 µg/mL without S9 mix. The concentration range of spirodiclofen for the mutagenicity study was chosen according to the results of the cytotoxicity test with spirodiclofen. The solvent was DMSO. Ethylmethanesulfonate (EMS) was used as a positive control without S9 mix, and dimethylbenzanthracene (DMBA) as a positive control with S9 mix.

Rationale for test conditions:

Test concentrations were selected on the basis of a preliminary cytotoxicity assay.

Evaluation criteria:

Normally, an assay is only considered acceptable for evaluation if the following criteria are met. However, the study conclusions are to be based upon the Study Director's evaluation and interpretation of the data.
- The activation and nonactivation assays are repeated at least once independently.
- The average cloning efficiency of the negative and vehicle controls should be at least 50%. Assays below 50% cloning efficiency will be unacceptable.
Cytotoxicity is determined after treatment with the test substance by the assay parameter "Survival to Treatment".
The highest test substance concentration should produce a low level of relative survival (0 - 30%) or should be the first concentration where insolubility occurs. The survival at the lowest concentration should approximate the negative control.
The background mutant frequency (average value for vehicle controls) in a trial should not exceed 25x10e6 cells. Assays with higher spontaneous mutant frequencies are not necessarily invalid, however, if all other criteria are fulfilled. An experimental mutant frequency is considered acceptable only if the absolute cloning efficiency is 10% or greater. Mutant frequencies for at least four concentrations of the test substance are routinely determined in each assay.Mutant frequencies are normally derived from sets of 8 dishes per parallel-culture of each dose level. To allow for contamination losses, an acceptable mutant frequency can be calculated from a minimum of 5 dishes. The positive control must induce an average mutant frequency of at least three times that of the vehicle control. An assay will be considered positive if a dose-dependent, significant and in parallel cultures reproducible increase in mutant frequency is observed. It is desirable to obtain this dose-relationship for at least 3 doses. To be significant, the mutagenic response to the substance should be at least approximately two to three times that of the highest
negative or vehicle control values.

Statistics:

The statistical analysis relies on the mutation frequencies rather than on individual plate counts which are submitted to a weighted analysis of variance as well as to a weighted recursive regression, both with Poisson derived weights. Mutation frequencies based on less than 5 plate counts are very uncertain estimates of the true mutation frequencies and provide no basis for a rational estimation of the variance. Therefore, such values are not included in the statistical analysis. If the relative population growth in an experiment falls below 10%, the corresponding mutation frequency is discarded. The two mutation frequency values obtained per group in the standard HPRT assay are, although somewhat related, considered as independent measurements thus increasing the power of the statistical tests applied. Since the protocol of the HPRT assay requires at least two replicates, the overall analysis per type of metabolic activation is the most important one for classifying substances into mutagens and non-mutagens. However, separate analyses will be run for each assay in order to examine the consistency of the results. All groups are included in the weighted analysis of variance followed by pairwise comparisons to the vehicle
control on a nominal significance level of a= 0.05 using the Dunnett test. The regression analysis part is performed on the basis of the actual dose levels thereby omitting the positive, negative and vehicle controls. If there is a significant increase of the mutation frequency with dose (a = 0.05) in the main analysis the highest dose group will be dropped and the analysis will be repeated until p > 0.05. Dose levels eliminated in that way are flagged correspondingly.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Under non-activation conditions two trials were performed. A dose-related decrease was observed both in relative survival and relative population growth. Immediately after treatment, virtually all cells were lost at a test substance concentration of 20 ug/mL in both experiments and at a concentration of 15 ug/mL in one experiment due to cytotoxicity. There was a slight increase of the mutation frequency in one culture at concentrations of 6 ug/mL, 8 ug/mL and 10 ug/mL
These increases were considered to be of no biological relevance since they could not be reproduced in the parallel treated culture or the second trial. The overall statistical analysis of the two trials revealed no statistically significant increase in mutation frequency at any of the tested concentrations. Therefore, no biologically relevant and reproducible increase of the mutation frequency over the concurrent vehicle controls was evident. The positive control EMS induced a clear mutagenic effect in all experiments. Therefore, spirodiclofen was evaluated as non-mutagenic in the non-activation trial.

Under metabolic activation conditions, 2 trials were performed. In all experiments, high toxicities
were induced in that the treated cultures showed dose-related decreases in both relative survival to treatment and relative population growth. The slight increases of the mutation frequency in one culture at concentrations of 10 and 20 ug/mL were considered to have no biological relevance since these increases were not reproducible in the parallel treated culture or the second trial. The joint statistical assessment of the 2 trials resulted in a statistically significant increase of the mutation frequency over the concurrent vehicle controls at concentrations of 10, 20 and 50 ug/mL. These statistical significances are considered to be of no biological relevance, since mutation rates at these concentrations do not represent a mutagenic response according to the evaluation criteria. In addition, these significances occurred in the absence of any dose-relationship. Therefore, no biologically significant increases in mutant frequency could be observed in treated groups compared to the concurrent vehicle controls according to the evaluation criteria. The positive control substance DMBA showed a clear mutagenic effect in all trials. Therefore, spirodiclofen was evaluated as non-mutagenic with metabolic activation in this test system.

Summary of results without metabolic activation

Concentration (µg/mL)	-S9
	Experiment 1		Experiment 2
	RTG	MF	RTG	MF
0 (untreated)	62.9, 70.4	0.5, 0.6	81.1, 66.6	2.7, 1.5
0 (DMSO	100, 100	1.0, 2.3	100, 100	3.5, 2.1
4	71.2, 95.8	1.0, 1.2	71.8, 101.1	3.7, 3.1
6	81.9, 107.4	5.5, 1.5	69.6, 95.8	3.5, 0.6
8	48.5, 104.3	5.8, 2.0	73.2, 124.6	1.6, 2.6
10	16.6, 32.9	9.7, 1.7	69.3, 83.7	3.1, 0.6
15	-	-	8.1, 5.9	0.6, 0.5
20	-	-	-	-
EMS	24.9, 47.1	435.6, 595.1	25.8, 21.9	575.5, 665.6

RTG: relative total growth

MF: mutation frequency (mutant colonies/10⁶ cells)

 

Summary of results with metabolic activation

Concentration (µg/mL)	+S9
	Experiment 1		Experiment 2
	RTG	MF	RTG	MF
0 (untreated)	107.6, 90.7	3.2, 5.1	112.9, 91.5	6.1, 4.5
0 (DMSO	100, 100	0.9, 2.4	100, 100	1.3, 0.5
10	132.6, 75.7	5.1, 10.7	82.1, 99.0	2.6, 1.9
20	110.2, 92.6	11.4, 6.5	65.5, 85.9	2.2, 2.0
40	88.8, 87.2	1.0, 6.6	62.2, 74.6	1.5, 4.4
50	71.6, 57.8	6.3. 4.5	55.3, 64.8	7.8, 2.5
60	84.3, 45.4	4.8, 4.8	42.6, 24.6	3.4, 0.4
80	34.8, 30.9	7.2, 2.3	-
DMBA	57.3, 43.7	131.3, 104.6	48.6, 61.2	167.7, 78.8

RTG: relative total growth

MF: mutation frequency (mutant colonies/10⁶ cells)

 

 

 

 

Conclusions:

Spirodiclofen was considered to be non-mutagenic in the V79-HPRT Forward Mutation assay with
and without metabolic activation.

Executive summary:

Spirodiclofen was tested for mutagenic effects at the hypoxanthine-guanine phosphoribosyl transferase locus (forward mutation assay) in V79 cell cultures in vitro with concentrations of up to 80 μg/mL (+S9) and up to 20 μg/mL (-S9). The concentration range for the mutagenicity study was chosen according to the results of a preliminary cytotoxicity test. The solvent was DMSO.  Ethylmethanesulfonate (EMS) was used as a positive control (-S9);  dimethylbenzanthracene (DMBA) as a positive control (+S9).  Spirodiclofen induced decreases in survival to treatment and decreases in relative population growth.  These results revealed a significant concentration-related cytotoxicity with and without metabolic activation. There was no significant concentration-related or reproducible increase in the mutant frequency above that of the negative controls. The positive controls demonstrated a good sensitivity of this assay.  Spirodiclofen was therefore considered to be non-mutagenic in the V79-HPRT Forward Mutation assay, with and without metabolic activation.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: 92/69/EEC

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

LT2: Reversion to histidine independence

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Cytokinesis block (if used):

Not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Concentrations of up to and including 5000 µg/plate (limit concentration)

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

cumene hydroperoxide

other: nitrofurantoin, 4-nitro-1,2-phenylene diamine and 2-aminoanthracene.

Remarks:

2-AA was used as the sole positive control for all strains (+S9)

Details on test system and experimental conditions:

The test material was tested in this Salmonella/microsome assay at concentrations of up to and including 5000 µg/plate. The solvent was DMSO. The Salmonella strains were the histidine-auxotrophic strains TA 1535, TA 100, TA 1537, TA 98 and TA 102. Sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine, cumene hydroperoxide and 2 aminoanthracene were used as positive controls.

Key result

Species / strain:

S. typhimurium TA 102

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:

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:

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:

not applicable

True negative controls validity:

not applicable

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:

not applicable

True negative controls validity:

not applicable

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:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

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:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

Spirodiclofen was negative in this Ames test in five S. typhimurium strains in the presence and absence of metabolic activation.

Executive summary:

Spirodiclofen was investigated for point mutagenic effects using the plate incorporation and the preincubation modification of the Ames test using concentrations of up to and including 5000 ug/plate on five Salmonella typhimurium histidine-auxotrophic strains TA 1535, TA 100, TA 1537, TA 98 and TA 102.  Concentrations of up to and including 5000 ug/plate did not cause any bacteriotoxic effects; total bacteria counts remained unchanged and no inhibition of growth was observed.  In both trials, no evidence of mutagenic activity was seen. No biologically relevant increase in the mutant count, in comparison with the negative controls, was observed.  The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine, cumene hydroperoxide and 2-aminoanthracene showed a marked and biologically relevant increase in mutant colonies compared to the corresponding negative controls.  Therefore, spirodiclofen was considered to be non-mutagenic without and with metabolic acitvation in the plate incorporation as well as in the pre-incubation modification of the Ames test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

A GLP- and guideline- (OECD 474) compliant mouse bone marrow micronucleus assay is available for spirodiclofen.  The study used intraperitoneal dosing (with evidence of target tissue exposure), and reports a negative result.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

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

Qualifier:

according to guideline

Guideline:

other: 92/69/EEC

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Version / remarks:

not specified

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

Standard laboratory species/strain

Sex:

male/female

Route of administration:

intraperitoneal

Vehicle:

0.5% aqueous Cremophor emulsion

Details on exposure:

Male and female mice received a single intraperitoneal administration of 800 mg/kg bw.

Duration of treatment / exposure:

Single intraperitoneal injection, with sampling at 16, 24 and 48 hours.

Frequency of treatment:

Single dose

Post exposure period:

The femoral marrow of groups was prepared 16, 24 and 48 hours after administration. Negative and positive controls were sacrificed 24 hours after administration.

Dose / conc.:

800 mg/kg bw/day (actual dose received)

Remarks:

intraperitoneal administration

No. of animals per sex per dose:

20/sex

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide served as positive control; the dose was 20 mg/kg bw ip.

Tissues and cell types examined:

Femoral bone marrow: polychromatic erythrocytes (PCEs)

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

After single intraperitoneal administration of 800 mg/kg bw, mice showed the following signs until sacrifice: apathy, roughened fur, spasm and eyelids stuck together. Their feeding behaviour
was normal. One of forty treated animals died during the test period. No symptoms were recorded for the control groups. No animals died in these groups.

The ratio of polychromatic to normochromatic erythrocytes was clearly altered by treatment with spirodiclofen at all time points.

No biologically or statistically significant variations existed between the negative control and the groups treated intraperitoneally with 800 mg/kg bw spirodiclofen with respect to the
incidence of micronucleated polychromatic erythrocytes.

Summary of the mouse bone marrow micronucleus assay with spirodiclofen

Group	Time point
	16h		24h		48h
	NCE:PCE^1	MnPCE^2	NCE:PCE^1	MnPCE^2	NCE:PCE^1	MnPCE^2
Vehicle control	-	-	942	1.8	-	-
800 mg/kg bw	2501	1.4	2166	1.9	2035	1.9
Positive control	-	-	913	16.4	-	-

¹ number of NCEs per 1000 PCEs

² number of micronuclei/1000 PCEs

Conclusions:

This study showed no evidence of micronucleus formation in the bone marrow of mice, with evidence of target tissue exposure.

Executive summary:

Spirodiclofen was tested for clastogenic potential in the bone-marrow erythroblasts of male and female mice. Male and female mice received a single intraperitoneal administration of 800 mg/kg bw.  The femoral marrow of groups were prepared 16, 24 and 48 hours after administration. Negative and positive controls were sacrificed 24 hours after administration. Cyclophosphamide served as positive control, the dose was 20 mg/kg bw ip.  Spirodiclofen-treated mice displayed clinical signs including rough fur, apathy, spasms and eyelids stuck together; one mouse died. The PCE:NCE ratio was altered by treatment with spirodiclofen, demonstrating target tissue exposure.  The incidence of micronucleated was unaffected by treatment with spirodiclofen.  The positive control cyclophosphamide had a clear clastogenic effect, an increase in polychromatic erythrocytes with micronuclei, and thus proved the sensitivity of this test system.  Spirodiclofen was therefore not clastogenic under the conditions of this assay. Conclusion: This study did not reveal evidence of a clastogenic effect of spirodiclofen in an in vivo system in mice.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Negative results are reported in vitro for spirodiclofen in an appropriate battery of assays addressing gene mutation and chromosomal aberration endpoints.  A negative results is also reported in vivo in a mouse bone marrow micronucleus assay.  In the absence of any evidence for genetic toxicity, spirodiclofen does not require classification for germ cell mutagenicity according to the CLP criteria.