Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No reliable in vitro genetic toxicity study was available for the test substance; therefore a read-across approach with the pharmacologically active ingredient Imazalil was used to cover these endpoints.

Bacterial reverse mutation assay

2 key studies are used in a weight of evidence approach to cover this endpoint:

-        In a K2 GLP bacterial reverse mutation assay in  Salmonella typhimurium  (TA1535, TA1537, TA98 and TA100) and in  Escherichia coli  WP2uvrA strains performed according to a method similar to the OECD Guideline 471 (Watabe et al., 1992), the source substance Imazalil was concluded that the test substance was not mutagenic in the absence and presence of metabolic activation.

-        In a second K2 GLP bacterial reverse mutation assay in Salmonella typhimurium  (TA1535, TA1537, TA1538, TA98 and TA100) performed according to a method similar to the OECD Guideline 471 (Vanparys P, 1988), it was concluded that the source substance Imazalil, in the absence and in the presence (20 and 50 μl S9/plate) of a rat metabolic activation system, has no mutagenic properties towards the various Salmonella typhimurium strains under these test conditions.

In vitro chromosome aberration study

In a K2 in vitro chromosome aberration study in human lymphocytes (Vanparys P, 1990), performed according to a method equivalent to OECD Guideline 473, it is concluded that the source substance Imazalil, in the presence and absence of a rat liver metabolic activation system, has no clastogenic properties in human lymphocytes.

 

In vitro gene mutation study in mammalian cells

In a K2 in vitro gene mutation study in Chinese hamster lung fibroblasts (V79) using the HPRT gene, performed according to OECD Guideline 476 (Van Gompel J, 1995), it can be concluded that the source substance Imazalil the absence and in the presence of a rat liver metabolic activation system, has no mutagenic properties towards the V79 cells under the test conditions described.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1988-01-22 to 1988-02-05
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Out of the five strains tested, four are recommended by test guideline OECD 471
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Remarks:
However 4/5 recommended strains tested
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: ZR 23979 D8801
- Expiration date of the lot/batch: no data
- Purity test date: 1988-02-25

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored in a refrigerator in closed containers
- Stability of test item: known
- Stability of prepraration: not required as it is prepared for immediate use

OTHER SPECIFICS: /


Target gene:
Histidine locus (histidine-dependent S. typhimurium strains)
Species / strain / cell type:
S. typhimurium, other: TA1535, TA97, TA1538, TA98, TA100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
Not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (Aroclor 1254 induced rat liver metabolic activation system)
Test concentrations with justification for top dose:
The maximum final concentration for the dose range finding test was based on the solubility of the test item in DMSO (maximum recommended concentration for test item soluble in vehicle)
Dose-range finding test: 25, 50, 100, 250, 500, 1000, 2000, 3000, 4000 and 5000 μg/plate in TA100 with and without 50 µl S9/plate.

The top doses for the mutation experiments were selected based on the toxicity observed in the dose range finding test.
First study: 5, 10, 25, 50, 100, 250 and 500 μg/plate in TA97, TA98, TA1538, TA100 and TA1535 with and without 20 µl S9/plate
Repeated study: 5, 10, 25, 50, 100, 250 and 500 µg/plate in TA97, TA98, TA1538, TA100 and TA1535 with and without 50 µl S9/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide
- Justification for choice of solvent/vehicle: The test item was found to be soluble in DMSO up to a concentration of 5000 µg/plate. Upon mixing with water, a precipitation was found at the concentration of 1250 microgram test item / plate onwards. In spite of the precipitation, it was decided to test the test item in a preliminary range finding study up to a concentration of 5000 µg/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Without S9-mix; 5 microgram/plate (TA1538, TA98, TA97)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Without S9-mix; 1 microgram/plate (TA1535, TA100)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
With S9-mix; 1 microgram/plate (TA1538, TA98, TA97, TA1535, TA100)
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate incorporation
- without metabolic activation, 0.1 ml of the solvent control or positive control or test article concentration is introduced into a test tube, whereafter 2 ml of a histidine-biotin (0.5 mM) supplemented top-agar is added, followed by 0.1 ml of an overnight culture in nutrient broth (oxoid). The content of the tube is then mixed well with the vortex mixer and poured on to minimal glucose agar (Vogel-bonner E medium) petri dishes. The petri plates are then immediately tilted and swirled to ensure that the still molten top-agar is evenly distributed.
- with metabolic activation, 0.5 ml of the S9-mix is added to the test tubes after the overnight culture has been added. As quickly as possible after adding the S9-mix into the tubes, the content of the tube is mixed with the vortex mixer and poured on the plates. The petri dishes are then tiled and swirled to ensure proper distribution of the top-agar.
The plates are incubated in the dark at 37°C for 48 hours.
Top agar in top agar tubes was melted by heating to 45 ± 2°C.

DURATION
- Exposure duration: 48 h
- Selection time (if incubation with a selection agent): 48h (simultaneous with exposure)

SELECTION AGENT (mutation assays): Histidine (S. typhimurium histidine-dependent strains)

NUMBER OF REPLICATIONS: the plate incorporation tests are performed in triplicate

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertant colonies per plate and/or by a thinning or disappearance of the background bacterial lawn.
Evaluation criteria:
The number of the revertant colonies in the solvent control plates are taken as the background level of mutation for the assay.

According to Brusick reversion values are considered as positive if:
- the test article produces at one concentration level at least a twofold increase in the mean number of revertants with one of the strains TA97, TA98 or TA100, or a threefold increase in the mean number of revertants with one of the strains TA1535 or TA 1538.
- a dose effect relationship is observed
- these effects can be reproduced.
The above mentioned criteria can be applied in most cases. When equivocal results are obtained, more assays may be required, in orde to evaluate the mutagenic potential of the test article. If the test article is positive using one of two S9 concentrations per plate, repeated studies will be done using the optimum S9 concentration. If a test article produces a positive response in a single test that cannot be reproduced in additional runs, the initial positive test data loose significance.
Statistics:
No data
Species / strain:
S. typhimurium, other: TA1538, TA98, TA97, TA1535, TA100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Upon mixing with water, a precipitation was found at the concentration of 1250 microgram/plate onwards.
- Precipitation:
First study: no precipitation was observed in any of the conditions tested
Repeated study: no precipitation was observed in any of the conditions tested

RANGE-FINDING/SCREENING STUDIES: In the preliminary range finding test, the test item was tested at a concentration range of 25 to 5000 microgram/plate in the absence and presence of 50 µl S9/plate in the tester strain TA100. At the concentration of 250 µg/plate in the absence and presence of metabolic activation, pinpoints were observed indicating a toxic action of the test imte towards the strain TA100. Severe bacteriotoxic effects (absence of bacterial background) were observed at the concentration of 1000 µg/plate onwards in the presence and absence of metabolic activation. On basis of the severe toxic effects observed, it was decided to test the test item in the main study up to 500 µg/plate. In this preliminary study, no significant incraese in revertant colonies could be evidenced with any of the concentration in the absence as well as in the presence of rat liver S9 mix.

HISTORICAL CONTROL DATA
The number of revertant colonies in the solvent control group for each of the Salmonella typhimurium strains in the absence as well as in the presence of rat liver S9-mix falls within the laboratory background historical range of revertant colonies.
The positive controls showed a significant increase in the number of revertant colonies indicating their mutagenic activity. However in the first experiment, the mutation frequencies measured in TA98 in the absence of S9 are just below the historical value control.
On basis of these findings, this study is considered acceptable for the evaluation of the mutagenic potential of the test item.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
With all the strains, bacteriotoxic effects (visualized by a thinning in the bacterial backgground lawn or occurrence of pinpoints or absence of a bacterial background) were found at the concentration of 250 µg test item/plate onwards.

Main study

At concentrations of 5, 10, 25, 50, 100, 250 and 500 µg/plate the test item dit not reveal a significant increase in the number of revertant colonies, either in the presence or absence of a rat liver metabolic activation system.

Repeated study:

No mutagenic activity of the test item was observed confirming the findings observed in the first study. Higher concentrations of S9 (5 microliter/plate) did not induce an increase in the number of revertant colonies.

Conclusions:
Based on the lack of increase of the reversion rate, it can be concluded that the test item, in the absence and in the presence (20 and 50 µl S9/plate) of a rat metabolic activation system, has no mutagenic properties towards the various Salmonella typhimurium strains under these test conditions.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1992-03-09 to 1992-03-19
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Remarks:
no historical data tables provided in the report
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: ZR023979G3C941
- Expiration date of the lot/batch: not indicated
- Purity test date: not indicated

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature and protection from light
- Stability under test conditions: not indicated
- Solubility and stability of the test substance in the solvent/vehicle:
- in water: 0.018%
- in DMSO: more than 5%
- in acetone: 50%
- in methanol: more than 50%

Solutions of test substance were freshly prepared just before each experiment.


Target gene:
Histidine locus (histidine-dependent S. typhimurium strains); Tryptophan locus (tryptophan-dependent E. coli strains)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
Not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (phenobarbital (PB) and 5, 6-Benzoflavone (BF) induced rat liver metabolic activation system)
Test concentrations with justification for top dose:
Dose-range finding test: 4.9, 19.5, 78.1, 313, 1250 and 5000 μg/plate in TA98, TA100, TA1535, TA1537 and WP2uvrA with and without S9-mix.

The top doses for the mutation experiments were selected based on the toxicity observed in the dose range finding test.
Main test: 9.8, 19.6, 39.1, 78.3, 156.5 and 313 μg/plate in TA98, TA100, TA1535, TA1537 and WP2uvrA with and without S9-mix.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide
- Justification for choice of solvent/vehicle: not indicated.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide
Remarks:
Without S9-mix; 0.01 μg/plate (TA100, WP2uvrA); 0.1 μg/plate (TA98)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9-mix; 0.5 μg/plate (TA1535)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9-mix; 80 μg/plate (TA1537)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S9-mix; 0.5 μg/plate (TA98); 1 μg/plate (TA100); 2 μg/plate (TA1535, TA1537); 10 μg/plate (WP2uvrA)
Details on test system and experimental conditions:
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): not specified
- Test substance added usiong preincubation method

The top agar (0.6% Agar, 0.5% NaCl) was melted by heating and mixed with sterile of 0.5mM L-Histidine/D-Biotin solution for the S. typhimurium TA strains in the ratio of 1 to 10, likewise mixed with sterile 0.5 mM-Tryptophan solution for the E. coli WP2 uvrA strain.
For test without metabolic activation, 0.1 ml of test substance solution, 0.5 ml of 0.1 M Na-phosphate buffer (pH 7.4) solution and 0.1 ml of the each fresh bacterial culture wer put into a test tube, and mixture was preincubated for 20 minutes in a water bath at 37°C. For test with metabolic activation, 0.5 ml of S9M-mix was added to above test tubes as for 0.1 M Na-phosphate buffer, and at test tube was preincubated in above same conditions. 2 ml of top agar were amixed with a preincubated mixture, and contents of each tube were mixed and poured over the surface of a minimal glucose agar plate. Overlay agar was allowed to solidify.
All plates were incubated for 48hrs. at 37°C and then all plates were examined for inhibition of bacterial growth using a stereomicroscope. Afterwards, the number of revertant colonies were counted.

DURATION
- Exposure duration: 48 hr
- Selection time (if incubation with a selection agent): 48h (simultaneous with exposure)

SELECTION AGENT (mutation assays): Histidine (S. typhimurium histidine-dependent strains); Tryptophan (E. coli tryptophan-dependent strains)

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): test groups and positive controls: duplicate; solvent control: triplicate
- Number of independent experiments: 2

DETERMINATION OF CYTOTOXICITY
- Method: inhibition of bacterial growth
Rationale for test conditions:
not indicated
Evaluation criteria:
The criteria for determining a positive result was a significant dose-related increase in the number of revertants and the detection of a reproducible and significant positive response (increase over 2 times in number of revertants compared with solvent control) for at least one of the test points on two main tests.
Statistics:
A statistical analysis didn't use for evaluation
Species / strain:
S. typhimurium, other: TA98, TA100, TA1535, TA1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test item was observed to be insoluble in water at 0.018 %
- Precipitation: No precipitation was observed in any of the conditions tested

RANGE-FINDING/SCREENING STUDIES: In the dose-range finding test, the test item was tested at a concentration range of 4.9 to 5000 μg/plate in the absence and presence of S9-mix in the tester strains TA98, TA100, TA1535, TA1537 and WP2uvrA. Toxicity was observed at 313 µg/plate and above in all tester strains; therefore the highest dose tested in the main test was 313 µg/plate.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%): not reported

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Main test: Bacteria toxicity was observed from 313 μg/plate with metabolic activation, and from 313 or 156.5 μg/plate without metabolic activation, in all tester strains.
Conclusions:
It was concluded that the test item Imazalil wasn't mutagenic in bacterial point mutation assay under the experimental conditions.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 1986-09-01 to 1986-10-20
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Study performed equivalent/similar to OECD Guideline 473 (In vitro Mammalian Chromosome Aberration Test) and EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test) with several data reporting deficiencies and limited detail on methodology.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Remarks:
however low number of cells scored
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Remarks:
however low number of cells scored
GLP compliance:
no
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: D8601
- Expiration date of the lot/batch: Not specified
- Purity: 98.3 (GS with IS)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Not specified
- Stability under test conditions: Known
- Solubility and stability of the test substance in the solvent/vehicle: Not required as prepared for immediate use

Target gene:
Not applicable
Species / strain / cell type:
lymphocytes: Human
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: primary culture of human lymphocytes
- Suitability of cells: according to guideline
- Normal cell cycle time (negative control): not specified

For lymphocytes:
- Sex, age and number of blood donors: 22 and 24 years
- Whether whole blood or separated lymphocytes were used: whole blood
- Whether blood from different donors were pooled or not: not specified
- Mitogen used for lymphocytes: used but not specified

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
0.5 mL of blood was added to 5 mL growht medium, which was composed of 4.35 ml Ham's F-10 medium, 0.54 ml bovine serum, 0.07 ml phytohemagglutinin and 0.04 ml penicillin-streptomycin. Cultures were incubated in UGB culture bottles at 37°C in a closed waterbath.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 rat liver homogenate
Test concentrations with justification for top dose:
According to the solubility of the test article, the following dose levels were initially chosen to determine the concentration which caused inhibition of the mitotic index:
23, 227, 455 and 909 µg of the test substance/ml culture without and with metabolic activation (S9-mix).

Based on the observations of the cytotoxicity study the following concentrations were chosen for the chromosome aberration test: 9, 36, 73 and 145 µg of the test substance/ml culture without and with metabolic activation.
Vehicle / solvent:
- Vehicle used: dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: The test substance was found to be soluble in DMSO at the required concentrations.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without metabolic activation (-S9); at 0.9 and 1.8 µg/ml
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation (+S9 -mix); at 91 and 182 µg/ml
Details on test system and experimental conditions:
METHOD OF APPLICATION: Approximately 48h after initiation, the cultures were exposed to the test and control articles as well in the absence as in the presence of S9-mix. The test and control articles were diluted in the vehicle (DMSO) immediately before use. The total DMSO volume in the cultures amounted to 100 µL (1.8% v.v)

DURATION
- Exposure duration: 48h Without S9-mix: 24h, With S9-mix: 2h
- Expression time (cells in growth medium): not specified no expression time
- Fixation time (start of exposure up to fixation or harvest of cells): Without S9-mix: 24h, With S9-mix: 2h

SPINDLE INHIBITOR : colchicine

STAIN: 1% lacto-orcein

NUMBER OF REPLICATIONS: 2 replicates per culture

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Fixed cells were dropped onto grease-free glass slides marked with the blind code. Four slides were prepared per culture and allowed to dry for at least 48 hours at room temperature. the slides were stained overnight with 1% lacto-orcein and mounted with a cover-slip.

NUMBER OF CELLS EVALUATED: the mitotic index of a culture was determined by counting the number of metaphases per 1000 cells.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 50 methaphase spreads per randomly coded culture; i.e. a total of 100 metaphases per concentration group. Only metaphases containing a minimum of 45 and a maximum of 47 centromers were scored.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: No
- Determination of endoreplication: NO


Evaluation criteria:
A test article is considered positive (clastogenic) in the chromosome aberration test if it induced in one of the test concentrations a statistically significant (P<= 0.05) increase in the number of cells with chromosome aberrations.
A test article is considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statiscally significant increase in the number of cells with chromosome aberrations.
Statistics:
The Fisher exact probability Test (one tailed P-values)
Species / strain:
lymphocytes: human
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:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Water solubility: No data
- Precipitation: No data

RANGE-FINDING/SCREENING STUDIES:
At the concentration of 227 µg/ml, the test substance induced a slight hemolysis and a 96% and 82% reduction in mitotic index respectively in the absence and in the presence of a metabolic activation system. At the concentration of 23 µg of the test substance/ ml culture, a 4% and 21% reduction in mitotic index was found respectively in the absence and presence of a metabolic activation system.

STUDY RESULTS
- Concurrent vehicle negative and positive control data
The positive control chemicals both produced statistically significant increases in the frequency of aberrant cells; Therefore it was assumed that the test conditions were optimal and the metabolic activation system functioned properly.
The number of cells with chromosome aberrations (gaps exluded and included) found in the negative and solvent controls were considered acceptable. Although older donors were used, the age of the donors had no influence on the number of cells with chromosome aberrations. If one can not excluse quantitative differences between different donors there are no reasons to believe that different healthy donors would react in a qualitative different manner.

Chromosome aberration test (CA) in mammalian cells:
- Results from cytotoxicity measurements:
o For lymphocytes in primary cultures: mitotic index (MI):
At the concentration of 145 µg/ml culture, the test item was strongly cytotoxic as only 3 metaphases were found per 1000 cells in the presence of ral liver S9 -mix.
At the concentration of 73 µg of the test item /ml culture, nearly 60% reduction in the mitotic index was found in the absence of a metabolic activation system, whereas no reduction at all was found in the presence of S9 -mix.

- Genotoxicity results
o At the concentration of 9, 36, and 73 µg/ml culture, the test item did not induce a significant and biological increase in the number of cells with chromosome aberrations (gaps excluded and included), neither the presence nor in the abscence of a rat liver metabolic activation system.
o At the concentration of 145 µg of test substance/ml culture, only 23 metaphases could be analysed in cultures incubated with rat liver S9 -mix (due to the strong cytotoxic effects). No increase in cells with chromosome aberrations was found among these 23 metaphases analysed of the 145 µg/ml group concentration.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: no data
- Negative (solvent/vehicle) historical control data: no data
Conclusions:
The test was performed up to the limit of toxicity. Decreasing concentration levels from this toxic level provided adequate number of data points to ensure that any possible dose-response would have been detected.
Based on the lack of increase in the number of cells with chromosome aberrations, it is concluded that the test item, in the presence and absence of a rat liver metabolic activation system, has no clastogenic properties in human lymphocytes.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1994-11-28 to 1995-02-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
Adopted April 4, 1984
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Annex V of the EEC Directive 88/302/EEC, part B: Methods for the determination of toxicity; "Other Effects-Mutagenicity: in vitro Mammalian Cell Gene Mutation Test".
Version / remarks:
Adopted May 30, 1988
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: in vitro mammalian cell gene mutation test using the Hprt gene
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: ZR023979BEB062
- Expiration date of the lot/batch: No data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: No data
- Stability under test conditions: stable in solution for at least 7 days
- Solubility and stability of the test substance in the solvent/vehicle: soluble in DMSO and stable in solution for at least 7 days

Concentrations were made immediately before use.
Only freshly prepared formulations were used. Stability was determined before initiation of the study and a reserve sample from the batch is retained for a period of minimum 5 years in the analytical department. A concentration and stability determination on the highest concentration was done during the dose range finding tested. A concentration determination on the highest concentration was done during the first and repeat mutation test.
Target gene:
Hypoxanthine-guanine PhosphoRibosyl Transderase (HPRT) locus
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: obtained from ICN-Flow and stored in liquid nitrogen at the Cellbank (Department of Virology)
- Suitability of cells: shown to be a sensitive indicator for mutagenic activity of a broad range of chemical classes.
- Normal cell cycle time (negative control): not specified

For cell lines:
- Absence of Mycoplasma contamination: not specified
- Number of passages if applicable: not specified
- Methods for maintenance in cell culture: not specified
- Cell cycle length, doubling time or proliferation index: not specified
- Modal number of chromosomes: not specified
- Periodically checked for karyotype stability: not specified
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: not described
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (rat liver metabolic activation system induced by Aroclor 1254)Type and composition of metabolic activation system:
- source of S9 : prepared from adult male Wilstar rats supplied by the laboratory's permanent non-inbred colony.The rats were injected intraperitoneally with a polychlorinated biphenyl mixture, Aroclor 124 at 500 mg/kg.
- method of preparation of S9 mix: The livers were prepared according to the methods given by Maron and Ames. The preparation of the liver S9 fraction was carried out at 0-4°C under steril conditions. The livers were aseptically removed, weighted and washed in chilled KCl. The livers were then homogenized. The homogenete was centrifuged at 9000 g whereafter the supernatant (S9 fraction) was transfered into sterile vials and stored below -70°C.
- concentration or volume of S9 mix and S9 in the final culture medium : 1%
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): checked for bacterial contamination. Quality determined by incubating the S. typhimurium strains TA98 and TA100 with increasing concentrations of S9 and a constant dose (20 µg/plate) of 2-aminofluorene.
Test concentrations with justification for top dose:
Before performing the main forward mutation test, the non-inhibitory concentration level was determined in a range finding study.
The highest dose tested in the dose range finding study was determined by the solubility of Imazalil in DMSO.
Dose range finding: 5, 10, 25, 50, 100, 250, 500, 750, 1000, 1250 µg/ml

The dose levels for the main study were selected based on the cytotoxicity observed in the dose range finding test:
First study: 10, 20, 40, 60, 80, and 100 µg/ml

A repeated study was performed with more narrow concentration intervals at the cytotoxic concentration levels:
Repeat study: 20, 40, 60, 65, 70 and 80 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: in DMSO, Imazalil was found to be soluble up to the concentration of 500 mg/ml. Upon mixing withMilliQ water (100 µl DMSO with Imazalil into 10 ml MilliQ water = 1 %), Imazalil was found to be soluble up to the concentration of 125 mg Imazalil/ml DMSO.
- Justification for percentage of solvent in the final culture medium: not specified
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Without S9-mix, 1 µl/ml
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
With S9-mix, 5 µg/ml
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 3 petri dishes/concentration evaluated for cytotoxicity determination and cloning efficienty; 5 petri dishes/concentration evaluated for mutation frequency determination
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): not specified
- Test substance added in medium

TREATMENT SCHEDULE:
- Exposure duration/duration of treatment: 4 hours

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 7 days
- Selection time (if incubation with a selective agent): 7 days culture in selective medium (6-thioguanine)
- Fixation time (start of exposure up to fixation or harvest of cells): ca. 14 days
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: not specified
- Criteria for small (slow growing) and large (fast growing) colonies: not specified

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cloning efficiency

METHODS FOR MEASUREMENTS OF GENOTOXICIY : determination of mutation frequency

Evaluation criteria:
A test article is mutagenic only if:
- The minimum criterion demonstrating mutagenesis for any given treatment in a mutant frequency >= 2 times the vehicle control mutant frequency. However if the increase falls within the range of two times the historical vehicle control, the test item is not considered mutagenic.
- A dose- related increase in mutant frequency should be observed with this relation for at least 3 concentrations depending on the concentration steps chosen for the assay and the toxicity at which mutagenic activity appears.

A test article is non-mutagenic if:
- The minimum increase in mutant frequency is not observed for a range of applied concentrations that extends to toxicity causing 80% reduction in cloning efficiency or in the case of relatively nontoxic materials, a range of applied concentrations extending to the maximum of 5 mg/ml culture medium (or 5 µl/ml) or in the case of nontoxic insoluble materials, a range of applied concentrations extending to at least the solubility limit in culture medium.
Statistics:
no data
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Concentrations of 100 µg/ml of the test sustance and higher proved to be cytotoxic as shown by a complete loss of cells.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH and osmolality: not examined
- Possibility of evaporation from medium: no data
- Precipitation and time of the determination: no precipitation observed

RANGE-FINDING STUDY- CYTOTOXICITY
In the range finding study, the test item was tested at 5,10, 25, 50, 100, 250, 500, 750, 1000, and 1250 µg/ml culture medium in the absence and presence of a rat liver metabolic activation system (1% S9-mix). The concentration of 100 µg of test item/ml and higher proved to be cytotoxic, with or without a metabolic activation system as shown by a complete loss of cells. No cytotoxicity was observed at the lower concentrations. 100 µg of test item/ml was therefore selected as the highest acceptable concentration for the main study.

STUDY RESULTS
Concurrent vehicle negative and positive control data :
- The positive control ethylmethanesulfonate (EMS) produced a significant increase in mutation frequency, indicating its mutagenic activity.
- The positive control dimethylbenzanthracene (DMBA) produced a significant increase in mutation frequency.
- The number of colonies in selective culture medium of the vehicle control, in the absence of rat liver S9-mix falls within the accepted range.
- The number of colonies in selective culture medium of the vehicle control, in the presence or rat liver S9-mix falls also within the accepted range.

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible : none
- Statistical analysis; p-value if any: none

HISTORICAL CONTROL DATA - see tables in "additional information on results"

Mutation Assays

First study

Cloning efficiencies

- At 10, 20 and 40 µg imazalil/ml culture medium, with and without metabolic activation, the cloning efficiencies were comparable to those of the vehicle control.

- At 60 µg imazalil/ml culture medium,with and without metabolic activation, a reduction in clonning efficiency was observed.

- Higher concentrations of 80 and 100 µg imazalil/ml culture medium were cytotoxic and resulted in complete cell loss.

Plating efficiency

- At 10, 20, 40 and 60 µg imazalil/ml culture medium, with and without metabolic activation, the platting efficiencies were comparable to those of the vehicle control.

- Higher concentrations of 80 and 100 µg imazalil/ml culture medium were cytotoxic and resulted in complete cell loss.

Mutation frequency

No mutant frequencies above 2 times the range of the respective vehicle controls, were observed with any of the Imazalil concentrations, neither without nor in the experiment with metabolic activation.

Second repeated study

Cloning efficiency

- At 20 and 40 µg imazalil/ml culture medium,without metabolic activation, the cloning efficiencies were comparable to those of the vehicle control. The concentrations 60, 65 and 70µg imazalil/ml culture medium induced a dose-related increase in cytotoxicity. Higher concentrations of 80 µg imazalil/ml culture medium were cytotoxic.

- At 20 and 40 µg imazalil/ml culture medium,with metabolic activation, the cloning efficiencies were comparable to those of the vehicle control.

- At 60 µg imazalil/ml culture medium,with metabolic activation, proved to be cytotoxic.

- Higher concentrations of 65, 70, and 80 µg imazalil/ml culture medium resulted in complete cell loss.

Plating efficiency

- At 20, 40, 60, 65 and 70 µg imazalil/ml culture medium, without metabolic activation, the platting efficiencies were comparable to those of the vehicle control.Higher concentrations of 80 imazalil/ml culture medium was cytotoxic and resulted in complete cell loss.

- At 20, 40, and 60, µg imazalil/ml culture medium, with metabolic activation, the platting efficiencies were comparable to those of the vehicle control. Higher concentrations of 65, 70 and 80 imazalil/ml culture medium was cytotoxic and resulted in complete cell loss.

Mutation frequency

No mutant frequencies above 2 times the range of the respective vehicle controls, were observed with any of the Imazalil concentrations, in the experiment without metabolic activation. In the experiment with metabolic activation, the concentration of 20 µg Imazalil/ml culture medium showed a mutant frequency above two times the concurent vehicle control. However, this increase remained within the range of the historical control data for the vehicle control and no dose-related effects could be evidenced as no increase in the mutant frequency was obseved at 40 and 60 µg Imazalil/ml culture medium.

As in the first study in the presence of rat liver S9 -mix, no increase in mutant frequency was observed at the concentration of 20 µg Imazalil / ml culture medium, we may conclude that the increase observed in the repeat study must be considered as a fortuitous finding of no biological importance.

Historical control data

With metabolic activation (+S9)

Test article   Concentr.(µg/ml)   Mean    Std. dev.      

     Range limit

Min. Max.

 N

 Vehicle (DMSO)

 0

 15.6

 9.8

 0

 30

 19

 Pos. Ctrl.(DMBA)

 5

 321.5

 155.9

 105

 615  17

Without metabolic activation (-S9)

Test-article    Concentr.(µg/ml)  Mean  Std. Dev.

    Range limit

Min. Max.

 N
Vehicle (DMSO)  0  11.4  6.9  0  22 25 
 Pos.Ctrl.(EMS)  1  953.2  467  298  1865

 21

Conclusions:
These tests were performed at the limit of toxicity. Decreasing concentration levels from the highest acceptable concentration level provided adequate number of data points to ensure that any possible dose-response would have been detected. As all the tests satisfied the criteria for a valid test, no further testing was done.
Based on the lack of a biologically significant increase of the mutation frequency, it can be concluded that imazalil in the absence and in the presence of a rat liver metabolic activation system, has no mutagenic properties towards the V79 cells under the test conditions described in this report.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Data from the related substance imazalil base is used to cover this endpoint. The justification for read across is attached in IUCLID Section 13.
Reason / purpose for cross-reference:
read-across source
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Concentrations of 100µg/ml of the test sustance and higher proved to be cytotoxic as shown by a complete loss of cells.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Conclusions:
These test were performed at the limit of toxicity. Decreasing concentration levels from the highest acceptable concentration level provided adequate number of data points to ensure that any possible dose-response would have been detected. As all the test satisfied the criteria for a valid test, no further testing was done.
Based on the lack of a biologically significant increase of the mutation frequency, it can be concluded that imazalil in the abscence and in the prescence of a rat liver metabolic activation system, has no mutagenic properties towards the V79 cells under the test conditions desribed in this report.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
Data from the related substance imazalil base is used to cover this endpoint. The justification for read across is attached in IUCLID Section 13.
Reason / purpose for cross-reference:
read-across source
Species / strain:
S. typhimurium, other: TA98, TA100, TA1535, TA1537
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:
E. coli WP2 uvr A
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
Conclusions:
No reliable genetic toxicity study is available for Imazalil sulfate. Therefore, reliable data from the supporting substance Imazalil is used to cover this endpoint. Imazalil was judged as not mutagenic. The same is assumed for Imazalil sulfate. Justification for this read across approach is included in IUCLID section 13.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
Data from the related substance imazalil base is used to cover this endpoint. The justification for read across is attached in IUCLID Section 13.
Reason / purpose for cross-reference:
read-across source
Species / strain:
S. typhimurium, other: TA1538, TA98, TA97, TA1535, TA100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not applicable
Positive controls validity:
valid
Conclusions:
No reliable genetic toxicity study is available for Imazalil sulfate. Therefore, reliable data from the supporting substance Imazalil is used to cover this endpoint. Imazalil did not increase the number of revertant colonies of any strains tested, and was judged as negative. The same is assumed for Imazalil sulfate. Justification for this read across approach is included in IUCLID section 13.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Data from the related substance imazalil base is used to cover this endpoint. The justification for read across is attached in IUCLID Section 13.
Reason / purpose for cross-reference:
read-across source
Species / strain:
lymphocytes: Human
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
Conclusions:
No reliable genetic toxicity study is available for Imazalil sulfate. Therefore, reliable data from the supporting substance Imazalil is used to cover this endpoint.
The test was performed with Imazalil up to the limit of toxicity. Decreasing concentration levels from this toxic level provided adequate number of data points to ensure that any possible dose-response would have been detected.
Based on the lack of increase in the number of cells with chromosome aberrations, it is concluded that the test item, in the presence and absence of a rat liver metabolic activation system, has no clastogenic properties in human lymphocytes. The same is assumed for Imazalil sulfate. Justification for this read across approach is included in IUCLID section 13.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No reliable in vivo  genetic toxicity study was available for the test substance; therefore a read-across approach with the pharmacologically active ingredient Imazalil was used to cover this endpoint.

 

 In a K1 GLP in vivo micronucleus test performed according to test guideline OECD 474 (Vanparys P, 1988), the source substance Imazalil did not show any potential for inducing micronuclei in the erythrocytic system in male and female mice.

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
Study period:
1987-10-06 to 1987-10-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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)
Version / remarks:
as described in the Annex of EEC Directive 87/449
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian erythrocyte micronucleus test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: ZR 23979 D 8801
- Expiration date of the lot/batch: not specified
- Purity test date: 1987-12-03
- manufacturing date: 1987-06-17

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature in closed containers
- Stability under test conditions: not specified
- Solubility and stability of the test substance in the solvent/vehicle: The stability of the test article is determined before the initiation of the study and a reserve sample from each batch of the test article is retained for a period of minimum 5 years in the analytical department.

FORM AS APPLIED IN THE TEST: suspended in propylene glycol
Species:
mouse
Strain:
Swiss
Details on species / strain selection:
The species and strain is selected following review of available data on toxicity, pharmacology and/or pharmacokinetics and also because of the possibility to compare the recorded data with the historical controls of the laboratory.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Albino Swiss mice, supplied by own permanent non-inbred laboratory colony (established in 1956)
- Age at study initiation: 5 weeks
- Weight at study initiation: Males – 33 to 35 g ; Females: 31 to 35 g
- Assigned to test groups randomly: not indicated
- Fasting period before study: not indicated
- Housing: individually housed in a numbered macrolon cage
- Diet (e.g. ad libitum): "Huybrechts" pelleted diet, administered in self-raising hoppers, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 2 °C
- Humidity (%): 60 ± 10%
- Air changes (per hr): approximately 18 per hour
- Photoperiod (hrs dark / hrs light): 12h/12h

IN-LIFE DATES: no data
Route of administration:
oral: unspecified
Vehicle:
- Vehicle(s)/solvent(s) used: Polyethylene glycol
- Justification for choice of solvent/vehicle: not indicated
- Concentration of test material in vehicle: not specified
- Amount of vehicle: 0.1 mL per 10 g bw
Details on exposure:
The solutions are only freshly prepared formulations. All animals received a single standard volume of 10 mL/kg body weight orally.
Duration of treatment / exposure:
one single administration
Frequency of treatment:
one single administration
Post exposure period:
And sampling of the bone marrow was done 24, 48 and 72 hours after dosing
Dose / conc.:
20 other: mg/kg bw
Remarks:
low dose; 24h, 48h and 72h sacrifice time
Dose / conc.:
80 other: mg/kg bw
Remarks:
medium dose; 24h, 48h and 72h sacrifice time
Dose / conc.:
320 other: mg/kg bw
Remarks:
high dose; 24h, 48h and 72h sacrifice time
No. of animals per sex per dose:
The 130 animals are divided into 13 groups, each of 5 males and 5 females
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide, sacrifice time 48h only.
Justification for choice of positive control(s): no
Route of administration: orally, once
Doses/ concentrations: dissolved in 0.4M Tartaric Acid + water, dose 40 mg/kg bw
As only freshly prepared formulations of the test article are used no stablilty of the preparations is required.
Tissues and cell types examined:
Bone marrow: polychromatic erythrocytes (PCE)
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: In order to determine the maximum tolerable dose level for the main micronucleus test, a preliminary toxicity test is performed with three or four dosage groups of 3 male and 3 female rats or mice.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): At the beginning of the treatment the animals (including the controls) were weighed and the individual volume to be administered was adjusted to the animals body weight. The animals received the test item, the vehicle or the positive control substance once. Sampling of the bone marrow was done 24, 48 and 72 hours after treatment.

DETAILS OF SLIDE PREPARATION: The animals were sacrificed by cervical dislocation. One femur per animal is dissected free and the bone marrow is rinsed out with fetal calf serum. After centrifugation a drop of the homogenous cell suspension is placed on a slide and spread as a smear with a blood smearing instrument. Slides are then coded to avoid subjective bias. Slides are left to dry and are fixed with methanol and stained with Wright on an automatic apparatus. Finally, the slides are permanently mounted.

METHOD OF ANALYSIS: The slides are examined by light microscopy (magnification 1000x). Aa total of one thousand polychromatic erythrocytes (PCE) are counted per animal and the number of micronucleated polychromatic erythrocytes are recorded. At the same time, the number of micronucleated normochromatic erythrocytes (NCE) are also recorded in the fields containing these 1000 polychromatic erythrocytes.
The ratio of polychromatic erythrocytes to normochromatic plus polychromatic erythrocytes is determined for each animal by counting a total of 1000 erythrocytes.
Evaluation criteria:
A test chemical is considered positive in the micronucleus test if it induces a statistically significant (p < 0.05) dose-related increase in the number of micronucleated polychromatic erythrocytes in the combined data for both sexes or in the data for male or female groups separately.

A test chemical is considered negative in the micronucleus test if none of the tested concentrations or sampling times showed a statistically significant (p < 0.05) increase in the number of micronucleated polychromatic erythrocytes either in the combined data for both sexes or in hte data for male or female groups separately.
The preceding cirteria are not absolute and other extenuating factors may enter into the final evaluation decision.
Statistics:
The significance of any inter-group differences is assessed by the Mann-Whitney U test.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 20 mg/kg bw; 80 mg/kg bw; 320 mg/kg bw
- Solubility: not reported
- Clinical signs of toxicity in test animals: In a preliminary toxicity study, the test item was administered once orraly to three male and three female mice at dose levels of 160, 320 and 640 mg/kg. All the males and females of the 640 mg test item / kg dosage group died at 24h after dosing. One male of the 320 mg test item / kg dosage group died at 48h after dosing.
- Evidence of cytotoxicity in tissue analyzed: The number of polychromatic erytghrocytes (PCE) to 200 PCE and normochromatic erythrocytes (NCE) examined was reduced in the 160 and 320 mg test item / kg dosage groups. The body weight of the 160 and 320 mg test item / kg treated males and females was also found to be strongly reduced.
On basis of the dose related increase in mortality, reduction in bonne marrow proliferation and decrease in body weight found with the 160, 320 and 640 mg/kg treated mice, 320 mg/kg bw was considered as the highest acceptable dose level for the main test.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): none
- Ratio of PCE/NCE (for Micronucleus assay): no increase in the number of micronucleated PCE was detected at any of the sampling times when male and female mice were dosed once with 20, 80 and 320 mg test item / kg orally. This means that up to a toxic dose level the test item did not show chromosome breaking or spindle poisoning activity resulting in the formation of micronuclei.
- Clinical signs of toxicity in test animals: In the 20 and 80 mg test item / kg dosage groups, all the animals survived the experimental period. In the 320 mg test item / kg dosage group two females of the 24h sampling time, one male and three females of the 48h sampling time and three males and one female of the 72h sampling time, died before sacrifice time.
The body weight of the 320 mg test item / kg treated animals was found to be significantly decreased (p<=0.01) at the 48 and 72h sampling times when compared to the solvent control group. The bone marrow proliferation of the 320 mg test item treated mice was also found to be significantly decreased (p <= 0.01 - p<=0.001) at the 48 and 72h sampling time.

CONTROLS
The cumulated laboratory background solvent control data on PCE of males and females show a mean of 1.1 micronucleated PCE / 1000 PCe with a range of 0-5/1000 PCE. At the sacrifice times, the rate of micronuclaeted PCE in the solvent control group falls within the laboratory background solvent control range .
The positive control shows at the 48h sacrifice time a significant increase (p<= 0.001) increase in the number of micronucleated PCE as a result of the chromosome breaking activity of cyclophosphamide.
Conclusions:
The test item did not show any potential for inducing micronuclei in the erythrocytic system in male and female mice.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Bacterial reverse mutation assay 

Two K2 key studies were selected to cover the bacterial reverse mutation endpoint.

• In the study conducted by Watabe et al. (1992), Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and Escherichia. coli WP2 uvr A were treated with solutions of Imazalil in DMSO using the pre-incubation method at the following dose levels: 9.8, 19.6, 39.1, 78.3, 156.5 and 313µg/plate, in the presence and in the absence of metabolic activation.

Bacteria toxicity was observed from 313 µg/plate with metabolic activation, from 313 or 156.5 µg/plate without metabolic activation, in all tester strains.
No precipitation was observed in any of the conditions tested.

No increase in the number of revertants was observed in any of the tester strains following exposure to Imazalil.

 

• In the study conducted by Vanparys (1988), five strains TA1535, TA1538, TA97, TA98 and TA100 of Salmonella typhimurium were tested in triplicate at the following dose levels: 5, 10, 25, 50, 100, 250 and 500 µg/plate in the presence and in the absence of a metabolic activation system (20 and 50 µg/plate S9-mix in the main and repeat experiments, respectively). The test item Imazalil was dissolved in DMSO.

With all the strains, bacteriotoxic effects (visualized by a thinning in the bacterial background lawn or occurrence of pinpoints or absence of a bacterial background) were found at the concentration of 250 µg test item/plate onwards.
No precipitation was observed in any of the conditions tested.

No increase in the number of revertants was observed in any of the tester strains following exposure to Imazalil.

 

 In vitro chromosome aberration study

Vanparys et al. (1990) investigated the effect of Imazalil on the induction of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system.

Imazalil was dissolved in DMSO and tested at concentrations of 9, 36, 73 and 145 μg/ml culture in the absence and presence of S9 -mix. At the maximum concentration of 145 μg test item/ml culture in the absence of rat liver S9 -mix, no chromosome analysis was carried out because of cytotoxicity whereas in the presence of S9 -mix only 23 metaphases could be analysed.

None of the tested test item concentrations induced a statistically and biologically significant increase in the number of cells with chromosome aberrations, neither in the presence nor in the absence of a rat liver metabolic activation system.

Positive control substances, mitomycin C in the absence of S9 -mix and cyclophosphamide in the presence of S9 -mix, both produced a statistically significant increase in the incidence of cells with chromosome aberrations.

It is concluded that the test item is not clastogenic in human lymphocytes under the experimental conditions described in this report.

 

In vitro gene mutation study in mammalian cells

Van Gompel et al. (1995) evaluated the ability of Imazalil to induce point and/or gene mutations in V79 cells in the HPRT gene according to OECD Guideline OECD 476.

V79 cells were exposed to the test item, dissolved in DMSO, at the following dose levels: 10, 20, 40, 60, 80, and 100 μg/ml. After an expression period of 7 days, V79 cells were cultured in 6-TG selective medium for 7 days. Mutant colonies were then fixed, coloured and counted. The test item did not induce a dose-related and/or biological significant increase in the mutation frequency at the HPRT-locus in the absence and presence of this metabolic activation system. The repeat study confirmed the results obtained in the first study, with the following dose levels:20, 40, 60, 65, 70 and 80 μg/ml.

Under the same conditions, ethylmethanesulfonate (EMS) and dimethylbenzanthracene (DMBA) produced a biologically significant increase in mutant frequency as compared to their vehicle control. This demonstrates the sensitivity of the assay and the metabolising activity of the S9 -mix.

It is concluded that the test item can be considered as not mutagenic to the HPRT-locus in the V79 test system under the experimental conditions described in this report.

 

In vivo mammalian somatic cell study

The induction of structural and/or numerical chromosome aberrations in bone marrow cells was assayed after single oral administration of the test item to young male and female mice (Vanparys, 1988). Male and female mice were dosed with 20, 80 and 320 mg test item/kg body weight at 24, 48 and 72h prior to preparation of the bone marrow. The high dose selection was determined, in a preliminary toxicity test, by evidence of clinical toxicity (death, weight loss), and depression of hematopoiesis.

A total of 1000 polychromatic erythrocytes per animal were screened for the presence of micronuclei. Structural and numerical chromosome aberrations were evaluated by the enumeration of the micronuclei in polychromatic and normochromatic erythrocytes. This experiment did not demonstrate any structural or numerical chromosome aberrations induced by the test item in the erythrocytes of mice bone marrow.

The dosing of male and female mice in a positive control group with 40 mg/kg cyclophosphamide orally led to an increase in the number of micronucleated polychromatic erythrocytes as a result of the chromosome breaking effect of cyclophosphamide.

Justification for classification or non-classification

Based on negative results in all in vitro and in vivo genetic toxicity tests with the source substance Imazalil and the criteria of the CLP Regulation (EC) 1272/2008, Imazalil sulphate should not be classified for mutagenicity.