cis-4-[3-(p-tert-butylphenyl)-2-methylpropyl]-2,6-dimethylmorpholine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

-Sup. Ames Test using TA1535, TA100, TA1537, TA98: Negative

-Key. Ames Test using TA1535, TA100, TA1537, TA98, E.coli WP2: Negative

-Key HPRT Assay on CHO cells: Negative

-Key. chromosome aberration study on CHL V79 cells: Slight increase in exchanges (+S9 mix), which was statistically not significant and not dose dependent: Equivocal with metabolic activation

-Sup. UDS-Assay on rat hepatocytes: Negative

-Sup. chromosome aberration study on human lymphocytes: Negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17. Jan. 1994 - 03. Mar.1994

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:

1984

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 Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Chinese Hamster Ovary Cells (CHO) K 1; ICN Flow Labs., Meckenheim, FRG
- Suitability of cells: Recommended by guideline

For cell lines:
- Absence of Mycoplasma contamination: Not specified
- Methods for maintenance in cell culture: Cells were routinely grown in monolayers at approx. 37°C in an incubator. For each subculture cells were trypsinized and reseeded at a density of approx. 100,000 or 300,000— 500,000 cells in 25 cm2 or 80 cm2 flasks respectively and covered with medium (approx.5ml medium for small flasks, approx. 10 ml for 80 cm2 flasks).
-Subcultivations: 2 subcultivations were performed per week Incubation conditions: approx. 37°C; approx.90% humidity; approx. 5% CO2
- Modal number of chromosomes: 18-22
- Periodically checked for karyotype stability: no
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
1. Ham's F12 medium, supplemented with 1% v/v glutamine (200 mM) and 10% v/v fetal calf serum (FCS); During exposure to the test substance Ham's F12 medium was used without FCS supplementation
2. Selection medium A ("HAT—medium"): Glutamine—and FCS-supplemented Ham's F12 medium containing per ml: 3.88x 10exp3 mg Thymidine13.61x 10exp3 mg Hypoxanthine 0.19x10exp3 mg Aminopterine
3.Selection medium B ("TG—medium"): Glutamine—and FCS—supplemented, Hypoxanthine—free Ham's F12 medium with 6-Thioguanine (TG) at a final concentration of 10 pg/ml. All media were supplemented with Penicillin (50IU/ml) and Strepto-mycin (50ug/ml).


37°C humidified atmosphere containing approx. 5% carbondioxide (incubator)

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: 5 male Sprague-Dawley Rats
- method of preparation of S9 mix: 500 mg Aroclor1254 solution in cornoil (20%w/v) per kg body weight was administered once intraperitoneally to 5 male Spraque—Dawley rats 5 days before sacrifice. At the 5th day the animals were sacrificed and the livers prepared. All the preparation steps for recovering the microsome enzymes were carried out using sterile solvents and vessels at a temperature of +4°C. The livers were weighed and washed in an equivalent volume of a 150mM CaCl2-solution (approx.1 ml per g wet liver), then cut into small pieces and homogenized in a 3-fold volume of 150 mM CaCl2 solution. After the homogenate had been centrifuged at 9,000g for 10 minutes at +4°C, the supernatant (S-9fraction) was deep frozen in 3 ml portions in dry ice and stored for a maximum of 12 months at -70 to -80°C
A sufficient amount of S-9Fraction was thawed to room temperature prior to each experiment. 3 parts of S-9 fraction were mixed with 7 parts of S-9 supplement of a stock solution
- concentration or volume of S9 mix and S9 in the final culture medium: 2ml S9-Mix + 7ml cell culture medium+ 1ml ml test substance

Test concentrations with justification for top dose:

Experiment 1 (without rat liver S-9 mix): 0;
0.1;
0.215;
0.464;
1;
2.15;
4.64;
10 µg/ml
Experiment1 (with rat liver S-9mix):0;
1;
2.15;
4.64;
10;
21.5;
46.4;
100µg/ml
Experiment2 (without rat liver S-9 mix):0;
0.25;
0.5;
0.75;
1;
2.5;
5;
7.5µg/ml
Experiment 2 (with rat liver S—9 mix):0;
10;
25;
50;
75;
100;
150;
200 µg/ml

In a range—finding cytotoxicity test cells were treated with Fenpropimorph at concentrations ranging from 0.05 to 100 µg/ml (without S-9 mix) and from 0.1 to 500 µg/ml (with S-9 mix). This resulted in marked cytotoxicity at concentrations > 5 µg/ml in the absence and > 100µg/ml in the presence of S-9 mix.

Vehicle / solvent:

1 % Ethanol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 2
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 500,000
- Test substance added in: medium

TREATMENT AND HARVEST SCHEDULE:
- Pretreatment of cells: During the week prior to treatment, spontaneous HPRT-deficient mutants were eliminated from the stock cultures by growing the cells for 3 - 4 days in HAT medium
- Exposure duration/duration of treatment: 4 hours



FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 1 week
- Selection time (if incubation with a selective agent): 1 week
- At the end of the expression period, 6 x 300,000 cells from each treatment group were seeded in six flasks with selection medium containing 6-thioguanine (10µg/ml) for 1 week
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 6 x ~300,000, A Coulter counter was used. The numbers obtained in the untreated controls were cross—checked in a Bürker chamber. Colony counting: Giemsa-stained colonies were scored visually
-Fixing and staining: Ethanol642/Giemsa

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Cloning efficiency

Rationale for test conditions:

In a range—finding cytotoxicity test cells were treated with Fenpropimorph at concentrations ranging from 0.05 to 100 µg/ml (withoutS-9 mix) and from 0.1 to 500 µg/ml (with S-9 mix). This resulted in marked cytotoxicity at concentrations > 5 µg/ml in the´absence and > 100µg/ml in the presence of S-9mix.

Evaluation criteria:

The criteria for a positive response are: Increases of the corrected mutation frequencies over the concurrent control values and over 15 mutants per 10exp6 clonable cells and/or the evidence of a dose—response relationship in the increase in mutant frequencies. Evidence of reproducibility of any increase in mutant frequencies. A statistically significant increase in mutantfrequenciesandtheevidenceofa dose-response relationship. Isolated increases of mutant frequencies above 15 mutants per 10exp6 clonable cells or isolated statistically significant increases in the lower concentrations without a dose relationship may indicate a biological effect, but are not regarded as sufficient evidence for mutagenicity.

Statistics:

The number of mutant colonies in each flask of the dose groups, the positive controls and the untreated controls was compared to that of the solvent control groups using the Fisher-Pitman-Test for the hypothesis of equal means. The independence of the 6 flasks within each group was assumed for this test. If the results were significant, labels (* for p < 0.05;** for p < 0.01) were printed with the group values (uncorrected mutant frequency) in the tables. The test was performed one—sided.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Reduced cell densities and reduced cloning efficiencies were found 18 - 20 hours after exposure to the test substance at concentrations >5 µg/ml in the experiments without S—9 mix and concentrations >100µg/ml in the presence of S—9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

It was concluded that Fenpropimorph did not demonstrate a mutagenic potential under the conditions used in this invitro gene mutation assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 1993 - March 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

1983

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Chinese Hamster lung Fibroblasts V79
- Suitability of cells: Advised by Guideline

For cell lines:
- Absence of Mycoplasma contamination: Checked
- Methods for maintenance in cell culture: Stock solutions were thawn at 37°C in a water bath and volumes of 0.5 ml were transferred into 25 cm2 plastic flasks which contain about 5.0 ml MEM (Minimal Essential Medium incl. glutamin), supplemented with 10% FCS (Fetal Calf Serum) and antibiotics. Cells were grown at 37°C with 5 % CO2 and > 90% humidity and subcultured twice weekly. Cell monolayers were suspended in culture medium after dispersion with 2.5 % trypsin solution (about 0.1 ml).
- Cell cycle length, doubling time: approx. 12 - 16 hours
- Modal number of chromosomes: 22
- Periodically checked for karyotype stability: yes
- Periodically ‘cleansed’ of spontaneous mutants: no

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: 5 male Sprague-Dawley Rats
- method of preparation of S9 mix: 500 mg Aroclor1254 solution in cornoil (20%w/v) per kg body weight was administered once intraperitoneally to 5 male Spraque—Dawley rats 5 days before sacrifice. At the 5th day the animals were sacrificed and the livers prepared. All the preparation steps for recovering the microsome enzymes were carried out using sterile solvents and vessels at a temperature of +4°C. The livers were weighed and washed in an equivalent volume of a 150mM CaCl2-solution (approx.1 ml per g wet liver), then cut into small pieces and homogenized in a 3-fold volume of KCl solution. After the homogenate had been centrifuged at 9,000g for 10 minutes at +4°C, the supernatant (S-9fraction) was deep frozen in 5 ml portions in dry ice and stored at -70 to -80°C
A sufficient amount of S-9Fraction was thawed to room temperature prior to each experiment. 3 parts of S-9 fraction were mixed with 7 parts of S-9 supplement of a stock solution
- concentration or volume of S9 mix and S9 in the final culture medium: 1ml S9-Mix + 5ml cell culture medium+ 50µl test substance

Test concentrations with justification for top dose:

According to the dose findings of pretests, 4 µg/ml without S-9 mix and 20 µg/ml with metabolic activation were selected as top doses. This selection was based primarily on the results of the mitotic index and the cell count. In general, three dose levels were assessed. The additionally selected higher and lower doses (marked with *) were planned to be evaluated only if the results obtained regarding cytotoxicity deviated from those obtained in the pretest.

-Experiment 1
Doses without S-9 mix
0.5µg/ml*
1.0µg/ml
2.0µg/ml
4.0µg/ml
5.0µg/ml*

Doses with S-9mix
2.5µg/ml*
5.0µg/ml
10.0µg/ml
20.0µg/ml
30.0µg/ml*

-Experiment 2
Doses without S-9mix
0.5µg/ml*
1.0µg/ml
2.0µg/ml
3.0µg/ml*

Doses with S-9mix
5.0µg/ml*
10.0µg/ml
20.0µg/ml
30.0µg/ml*

Vehicle / solvent:

- Vehicle used: Ethanol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Duplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 30,000-50,000
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 hours
- Harvest time after the end of treatment: 18 hours in Experiment 1 and 2
Additionally 28 hours for the two highest doses in experiment 2

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): 0.2µg colcemid/ml culture for 2-3 hours prior harvest
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): After incubation with colcemid, the culture medium was completely removed. For hypotonic treatment 5 ml of a 0.4% KCl solution which was at 37°C was added for about 20 minutes. Subsequently 5 ml of fixative (methanol:glacial acetic acid/3:1) which was at 4°C was added and kept for at least 15 minutes and then replaced. After about another 10 minutes fixative was replaced again and kept for at least 5 minutes at room temperature for complete fixation. The slides were taken out of the Quadriperm chambers, briefly dripped off and then rapidly passed through a Bunsen burner flame. The preparations were dried in the air and sub-sequently stained in a solution of Giemsa and Titrisol (15ml Giemsa, 185ml Titrisol pH7.2) for 10 minutes. After being rinsed twice in aqua dest. and clarified in xylene, the preparations were mounted in Corbit-Balsam.

- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): 2 Replicates, 100-200 cells scored

- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): If only a few cells were found or if the metaphases were of low quality, a chromosome analysis was not carried out.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
For determination of cytotoxicity additional cell cultures (using 25 plastic flasks) were treated in the same way as in the main experiment. Growth inhibition was estimated by counting the number of cells in the dose groups in comparison to the concurrent vehicle control at the end of the culture period

Rationale for test conditions:

According to pretesting,a dose-dependent growth inhibition was observed at about doses > 4 µg/ml without S-9 mix after a harvest time of 18 hours. With metabolic activation a slight decrease in the cell count was found at 30 µg/ml.

Statistics:

The statistical evaluation of the data was carried out using the MUCHAN program system (BASFAG). For each group the proportion of metaphases with aberrations was calculated. A comparison of each dose group with the vehicle control group was carried out using Fisher's exact test for the hypothesis of equal proportions. This test was Bonferoni Holm corrected over the dose groups separately for each time point and was performed one-sided.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Remarks:

Experiment 2

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

After 18 hours harvest time with S9-mix only the number of cells with exchanges were on the upper limit(10µg/ml and 30µg/ml)or exceeds the upper limit (20µg/ml)of the historical control range. No dose-dependent effect was observed

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

Chinese hamster lung fibroblasts (V79)

Remarks:

Experiment 1

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

With S9-mix the number of cells with exchanges exceeded the historcal control data (0-2.5%) with 5 % at 20 and 30 µg/ml. The elevation was not statistically significant

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

In summary, it can be stated that there is a slightly elevated number of exchanges in two experiments (+S9 mix) independent of each other even if statistically not significant.The total number of chromosomal aberrations, however, is not increased, i.e. breaks, deletions and fragments, which in the case of clastogenic active compounds are expected to be elevated too for theoretical reasons, are missing. A decision can not be made whether these exchanges are the result of a direct clastogenic effect or of an unknown indirect and non-genotoxic mechanism, and a definite judgement about the activity of the test substance can not be given.

Reference 3

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Unscheduled DNA synthesis

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

July 13, 1987 - June 8, 1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

No S-Phase block

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.5550 - Unscheduled DNA Synthesis in Mammalian Cells in Culture

Deviations:

yes

Remarks:

No S-Phase block

Principles of method if other than guideline:

- Principle of test: In vitro UDS-Assay with primary rat hepatocytes
- Short description of test conditions: Isolation of primary rat hepatocytes. Exposition of primary rat hepatocytes to the test article, vehicle and positive control (2-AAF) in vitro. Incorporation of 3H-Thymidin. Autoradiographic detection and counting of UDS-sites
- Parameters analysed / observed: UDS grains/nucleus; Survival at 21 hrs

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: In vitro UDS-Assay with primary rat hepatocytes

Species / strain / cell type:

hepatocytes:

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Freshly isolated rat hepatocytes from one adult male Fischer 344 rat (Charles River Breeding Laboratories)

MEDIA USED
- The cell cultures were established in Williams' Medium E supplemented with 5% fetal bovine serum, 2mM L-glutamine, 2.4µM dexamethasone, 90 U/mlpenicillin, 90 µg/ml streptomycinsulfate, and 140µg/ml gentamicin. After the establishment period, the dexamethasone and serum components were removed.

All cultures were maintained as Mono-Layers at about 37°C in a humidified atmosphere containing approximately 5% CO2

Metabolic activation:

without

Test concentrations with justification for top dose:

Eight treatments from 10µg/ml to 0.05µg/ml were prepared.
0.1 µg/ml; 0.25 µg/ml; 0.5 µg/ml; 1.0 µg/ml; 2.5 µg/ml; 5.0 µg/ml; 10.0 µg/ml

Concentrations from 50.0µg/ml to 1000µg/ml were lethal. At 25.0µg/ml the survival increased to 54 % of the solvent control and the cells had a round morphology unlike the flat morphology of the solvent controls. At 10.0µg/ml the cells appeared flatter and the survival was 84.1%. 5.00µg/ml treatment had a survival of 78.3% and the cellular morphology was closer to normal. Concentrations below 1.00µg/ml had morphologies and survivals similar to the solvent control.

Vehicle / solvent:

- Vehicleused: ethanol
- Justification for choice of solvent/vehicle: Tested for the best solubility ethanol vs. DMSO vs. cell culture medium

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicates
- Number of independent experiments: 1

METHOD OF TREATMENT/ EXPOSURE:

- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: Attachement period of 1.5 to 2 hours
- Exposure duration/duration of treatment: 20 hours


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Trypan-blue viable count of parallel replicates

Rationale for test conditions:

The test substance was insoluble in DMSO and cell culture medium in higher doses. Therefore ethanol was chosen as vehicle.
Concentrations from 50.0µg/ml to 1000µg/ml were lethal. At 25.0µg/ml the survival increased to 54 % of the solvent control and the cells had a round morphology unlike the flat morphology of the solvent controls. At 10.0µg/ml the cells appeared flatter and the survival was 84.1%. 5.00µg/ml treatment had a survival of 78.3% and the cellular morphology was closer to normal. Concentrations below 1.00µg/ml had morphologies and survivals similar to the solvent control.

Evaluation criteria:

The test material is considered active in the UDS assay at applied concentrations thatcause:
1.An increase in the mean net nuclear grain count to at least six grains per nucleus after subtraction of the concurrent negative control value, and/or
2.An increase in the percent of nuclei having six or more net grains to at least 10% of the analyzed population after subtraction of the concurrent negative control value, and/or
3.The percent of nuclei with twenty or more grains to reach or exceed of the analyzed population.

Species / strain:

primary culture, other: Hepatocytes from a male Fischer 344 rat

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Concentrations > 25µg/ml induced marked cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

The test material, Fenpropimorph, did not induce significant changes in the nuclear labeling of rat primary hepatocytes for an applied concentration range of 5.00µg/ml to 0.100µg/ml.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Salmonella typhimurium / Escherichia Coli reverse Mutation assay

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13. Sept. 2010 - 12.Apr.2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21. Jul. 1997

Deviations:

yes

Remarks:

For S9-Mix testing only 2-Aminoanthracene was used

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Male Wistar rats [Crl:WI(Han)] (200 - 300 g)
- method of preparation of S9 mix: TThe S9 fraction is prepared according to Ames et al. 1975 at BASF SE in an AAALAC-approved laboratory in accordance with the German Animal Welfare Act and the European Council Directive 86/609/EEC. At least 5 male Wistar rats [Crl:WI(Han)] (200 - 300 g; Charles River Laboratories Germany GmbH) received 80 mg/kg b.w. phenobarbital i.p. and β-naphthoflavone orally (both supplied by Sigma-Aldrich, 82024 Taufkirchen, Germany) each on three consecutive days. During this time, the animals are housed in Makrolon cages: central air conditioning with a fixed range of temperature of 20 - 24°C and a relative humidity of 30 - 70%. The day/night rhythm is 12 hours (light period from 6.00 - 18.00 hours and dark period from 18.00 - 6.00 hours). Standardized pelleted feed and tap water from bottles were available ad libitum. 24 hours after the last administration, the rats are sacrificed, and the livers are prepared using sterile solvents and glassware at a temperature of +4°C. The livers are weighed and washed in a weight-equivalent volume of a 150 mM KCl solution, then cut into small pieces and homogenized in three volumes of KCl solution. After centrifugation of the homogenate at 9 000 x g for 10 minutes at +4°C, 5 mL portions of the supernatant (so-called S9 fraction) are stored at -70°C to -80°C.
- concentration or volume of S9 mix and S9 in the final culture medium: 31.34g/l
0.5ml S9-Mix in 2,7 ml Agar/Medium
- quality controls of S9: Sterility controls have been performed

Test concentrations with justification for top dose:

33, 100, 333, 1000, 2750, 5500µg/plate

In agreement with the recommendations of current guidelines 5 mg/plate or 5 μL/plate are generally selected as maximum test dose at least in the 1st Experiment. However, this maximum dose will be tested even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate or > 5 μL/plate might also be tested in repeat experiments for further clarification/substantiation. In this study, due to the purity of the test substance 5.5 mg/plate was used as top dose in the first experiment.

Vehicle / solvent:

DMSO

- Justification for choice of solvent/vehicle:
Due to the limited solubility of the test substance in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2-AA) N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) 4-nitro-o-phenylenediamine (NOPD) 9-aminoacridine (AAC) 4-nitroquinoline-N-oxide (4-NQO)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- 3 test plates per dose or per control

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 0.1ml of a solution of approx 10exp8 bacteria/ml
- Test substance added in medium in agar (plate incorporation) or preincubation;

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 20min
- Exposure duration/duration of treatment: 48 – 72 hours


METHODS FOR MEASUREMENT OF CYTOTOXICITY
Toxicity detected by
-a decrease in the number of revertants
-clearing or diminution of the background lawn (= reduced his- or trp- background growth)
-reduction in the titer

Rationale for test conditions:

In agreement with the recommendations of current guidelines 5 mg/plate or 5 μL/plate are generally selected as maximum test dose at least in the 1st Experiment. However, this maximum dose will be tested even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate or > 5 μL/plate might also be tested in repeat experiments for further clarification/substantiation.

Evaluation criteria:

Generally, the experiment is considered valid if the following criteria are met:
• The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain
• The sterility controls revealed no indication of bacterial contamination.
• The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above (see Appendix 6).
• The titer of viable bacteria was ≥ 10exp8/mL.

The test substance is considered positive in this assay if the following criteria are met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system. A test substance is generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.

Species / strain:

E. coli WP2 uvr A

Remarks:

Standard plate test and preincubation test

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the standard plate test from about 100 μg/plate onward. In the preincubation assay from about 33 µg/plate onward.

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Remarks:

Standard plate test and preincubation test

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the standard plate test from about 100 μg/plate onward. In the preincubation assay from about 33 µg/plate onward.

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Remarks:

Standard plate test and preincubation test

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the standard plate test from about 100 μg/plate onward. In the preincubation assay from about 33 µg/plate onward.

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Remarks:

Standard plate test and preincubation test

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the standard plate test from about 100 μg/plate onward. In the preincubation assay from about 33 µg/plate onward.

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Remarks:

Standard plate test and preincubation test

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the standard plate test from about 100 μg/plate onward. In the preincubation assay from about 33 µg/plate onward.

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Under the experimental conditions chosen here, it is concluded that BAS 421 F (Fenpropimorph) is not a mutagenic test substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation.

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Report date Mar. 31. 1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

According to OECD Guideline 471. Testing was performed using four strains of Salmonella typhimurium: TA 1535, TA 100, TA 1537, TA98

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: At least 5 male Sprague-Dawley rats (200g-300g)
- Method of preparation of S9 mix: The S9-Mix was prepared according to Ames, B. N.; McCann, J.; Yamasaki, E.; Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mut. Res.,1, 347- 364(1975).
The S9-Mix is prepared freshly prior to each experiment. For this purpose, a sufficient amount of S-9 fraction is thawed at room temperature and 1 volume of S-9 fraction is mixed with 9 volumes of S-9 supplement (cofactors).
-Volume of S9 mix and S9 in the final culture medium: 0.5 ml S9-Mix + 110.2 ml Agar-Medium
-The following positive control substances are used to check the mutability of the bacteria and the activity of the S9-mix: 2.5µg 2-aminoanthracene (2-AA, dissolved in DMSO)

Test concentrations with justification for top dose:

1. Experiment:
Strains: TA1535, TA100, TA1537, TA98
Doses: 0, 20, 100, 500, 2500, 5000 µg/plate

2. Experiment:
Strains: TA98
Doses: 0, 5, 10, 20, 40, 80 µg/plate

3. Experiment:
Strains: TA1535, TA100, TA1537, TA98
Doses: 0, 6, 12, 25, 50, 100 µg/plate (TA 1535, TA100, TA1537)
0, 20, 100, 500, 2500, 5000 µg/plate (TA98)

4. Experiment (Preincubation):
Strains: TA1535, TA100, TA1537, TA98
Doses: 0, 6, 12, 25, 50, 100 µg/plate (TA 1535, TA100, TA1537)
0, 20, 100, 500, 2500, 5000 µg/plate (TA98)

A bacteriotoxic effect was observed depending on the test conditions from about 50µg/plate onward with TA1535, TA100 and TA1537. Using TA98 a weak bacteriotoxicity was observed at doses >=2500µg/plate.

A test substance precipitation was found from about 2500µg/plate onward

Vehicle / solvent:

- Vehicle: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO with and without S9-Mix

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: With S9-Mix: 2.5µg 2-Aminoanthracene dissolved in DMSO Without S9-Mix: 5 µg N-methyl-N'-nitroso-guanidine dissolved in DMSO 10µg 4-nitro-o-phenylendiamine dissolved in DMSO 100µg 9-aminoacridine chloride monohydrate dissolved in DMSO

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- 3 test plates per dose or per control

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): approx. >10^7 germs
- Test substance added in medium or in agar (plate test and preincubation)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 20 min 37°C
- Exposure duration/duration of treatment: 48 hours 37°C

METHODS FOR MEASUREMENT OF CYTOTOXICITY
-Observation of reduced His- background growth and decrease in number of his+ revertants

Rationale for test conditions:

A bacteriotoxic effect was observed depending on the test conditions from about 50µg/plate onward with TA1535, TA100 and TA1537. Using TA98 a weak bacteriotoxicity was observed at doses >=2500µg/plate.

A test substance precipitation was found from about 2500µg/plate onward

Evaluation criteria:

In general, a substance to be characterized as positive in the Ames test has to fulfill the follwoing requirements:
-Doubling of the spontaneous mutation rate (control)
-Dose-response relationship
-Reproducibility of the results

Species / strain:

S. typhimurium TA 98

Remarks:

Standard plate test and preincubation test

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A bacteriotoxic effect was observed depending on the test conditions from about 50 µg—500µg/plate onward with TA1535, TA100 and TA1537. Using TA98 a weak bacterio toxicity was observed at dose 2500µg/plate.

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Remarks:

Standard plate test and preincubation test

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A bacteriotoxic effect was observed depending on the test conditions from about 50 µg—500µg/plate onward with TA1535, TA100 and TA1537. Using TA98 a weak bacterio toxicity was observed at dose 2500µg/plate.

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Remarks:

Standard plate test and preincubation test

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A bacteriotoxic effect was observed depending on the test conditions from about 50 µg—500µg/plate onward with TA1535, TA100 and TA1537. Using TA98 a weak bacterio toxicity was observed at dose 2500µg/plate.

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Remarks:

Standard plate test and preincubation test

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A bacteriotoxic effect was observed depending on the test conditions from about 50 µg—500µg/plate onward with TA1535, TA100 and TA1537. Using TA98 a weak bacterio toxicity was observed at dose 2500µg/plate.

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

According to the results of the present study, the test substance Fenpropimorph is not mutagenic in the Ames test under the experimental conditions chosen here.

Reference 6

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: Chromosomal aberration in human lymphocytes

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

3. October 1981 - 5. March 1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Remarks:

Non-GLP No Guideline study

Qualifier:

no guideline followed

GLP compliance:

no

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: Human

Details on mammalian cell type (if applicable):

Fresh venous blood, drawn just before the beginning of the experiment from a healthy donor who had not ingested any drug for at least 20 days is used.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: liver microsomal fraction is obtained from the liver of Sprague Dawley CD (SD) BR male rats of about 200-250g which received the following enzyme inducers for 4 days according to the following scheme:
- Day 130 mg/kg i.p.Phenobarbital(H20)
- Day 260 mg/kg i.p.Phenobarbital(H20)
- Day 360 mg/kg i.p.Phenobarbital(H20) + 80 mg/kg i.p. Beta-Naphtoflavone(Cornoil)
- Day 460 mg/kg i.p. Phenobarbital(H20)
- Day 5 Sacrifice after 12 hours fasting

- method of preparation of S9 mix: After sacrifice, the livers are removed and chopped with sterile lancets and homogenized with Ultra turrax in a sterile solution of (solution/tissue ratio 1:3 v/w). The temperature of the container with the livers is kept at +4°C. The homogenate is then centrifuged for 10’ at 9000 rpm at +4°C . The supernatant is divided into fractions of varying volumes, placed in sterile screw-cap containers, and stored at -80°C.
- concentration or volume of S9 mix and S9 in the final culture medium: 1.25 ml of S9-Mix in 2.59 ml total volume
- quality controls of S9: Test in a Salmonella typhimurium reverse mutation test with 2-Anthramine. Determination of micromosmale proteins. Enzyme Assay with Aminopyrine-Demethylase activity

Test concentrations with justification for top dose:

The dose is determined from preliminary test for mitotic delay and mitotic index in cultures exposed to eleven log dose range of the test article 5000 µg/ml, 1666 µg/ml, 500 µg/ml, 166 µg/ml, 50 µg/ml, 16.6 µg/ml 5 µg/ml 1.66 µg/ml, 0.50 µg/ml, 0.16 µg/ml, 0.05 µg/ml
3 dilution doses of the initial solution of the test article are employed

In base of the results of the toxicity test, the following dose levels were Chosen:

Without S-9 mix: 1.66µg/ml 0.5 µg/ml, 0.16 µg/ml

With S9-mix: 50 µg/ml, 16.6 µg/ml, 5 µg/ml

Vehicle / solvent:

Ethanol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Duplicate
- Number of independent experiments:1

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 48 h preincubation with Phytohaemagglutinin
- Exposure duration/duration of treatment: 2h at 37°C
- Harvest time after the end of treatment (sampling/recovery times): 24 h

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): indicate the identity of mitotic spindle inhibitor used (e.g., colchicine), its concentration and, duration and period of cell exposure: 10exp-3M Colchicin for 3h at 37°C
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays):Cells are suspended for 15 min in a hypotonic KCL solution. After centrifugation cells are fixed with a 1:3 mixture of acetic acid and ethanol. The cells are transferred onto a slide and dried at the air. The cells are stained for 10 min in Giemsa at 3% Soerensen phosphate buffer.
- Number of cells spread and analyzed per concentration (number of replicate cultures and total number of cells scored): 100 well-spread metaphases were analysed using 2 slides per dose level.
- Criteria for scoring chromosome aberrations (selection of analyzable cells and aberration identification): Not specified


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Mitotic delay and mitotic index

Rationale for test conditions:

The dose is determined from preliminary test for mitotic delay and mitotic index in cultures exposed to eleven log dose range of the test article 5000 µg/ml, 1666 µg/ml, 500 µg/ml, 166 µg/ml, 50 µg/ml, 16.6 µg/ml 5 µg/ml 1.66 µg/ml, 0.50 µg/ml, 0.16 µg/ml, 0.05 µg/ml

The lowest concentration to reduce the mitotic index to 0-1% is used as the highest dose in the test.

3 dilution doses of the initial solution of the test article are employed

In base of the results of the toxicity test, the following dose levels were Chosen:

Without S-9 mix: 1.66µg/ml 0.5 µg/ml, 0.16 µg/ml

With S9-mix: 50 µg/ml, 16.6 µg/ml, 5 µg/ml

Statistics:

The statistical evaluation of the data is performed with the following tests:
a)Comparison of control (CHI square test) both with the standard mutagen and the treated preparations.
b)Control of the dose-effect correlation (linear regression in the case of aberration frequencies which differ significantly from the control.

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

-Erythrocyte Micronucleus Test: Negative

-Dominant lethal test: Negative

Link to relevant study records

Reference

Reference 1

Endpoint:

genetic toxicity in vivo, other

Remarks:

Dominant lethal test on male mice after single intraperitoneal administration

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

07. - 09.1978

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: A single dose of the test substance was intraperitoneally administered to male mice. These mice were mated with untreated females in a 1:1 relation and a 4-day mating rhythm over 12 mating intervals in order to record the total gametogenesis. At the time of treatment all stages of spermatogenesis, consecutively undergoing maturation, are present in the testes. Fractionated mating allows to record successively the stages of spermatogenesis.
- Parameters analysed / observed: Clinical signs and bw of treated male mice. The number implantations in total and the number of dead implantations is determined whereby an mortality in utero as compared to the spontaneous number, suggests induced, genetically caused lethal factors.

GLP compliance:

not specified

Type of assay:

other: Dominant Lethal Test

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SPF-Zucht Messrs. WIGA, Sulzfeld
- Age at study initiation: 14 - 16 weeks
- Weight at study initiation: 33.1 g (male animals), 25-30 g (female animals)
- Assigned to test groups randomly: yes
- Fasting period before study: not specified
- Housing: singly
- Diet: Altromin, ad libitum
- Water: water in bottles, ad libitum
- Acclimation period: not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): not specified
- Humidity (%): not specified
- Air changes (per hr): not specified
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: To: not specified

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: CMC
- Justification for choice of solvent/vehicle: not specified
- Concentration of test material in vehicle: 4.25 % (g/g); 2.125 % (g/g)
- Purity: 0.5 % CMC solution

Details on exposure:

intraperitoneal

Duration of treatment / exposure:

single intraperitoneal administration

Frequency of treatment:

once

Post exposure period:

48 days

Dose / conc.:

212.5 mg/kg bw (total dose)

Dose / conc.:

425 mg/kg bw (total dose)

No. of animals per sex per dose:

40 males (1 x 10 ml/kg bwt of CMC solution)
40 males (1 x 212.5 mg/kg bwt of test substance)
40 males (1 x 425 mg/kg bwt of test substance)

Control animals:

yes, concurrent vehicle

Positive control(s):

none

Tissues and cell types examined:

all animals:
- daily examination for externally recognizable clinical signs of toxicity

males:
- body weight was determined prior to the administration of the substance and prior to the beginning of a new mating interval
- after termination of the 12th mating interval the males of all test groups were sacrificed, dissected and macroscopically examined for pathological changes of the internal organs.

females:
- conception rate was calculated for each mating interval
- number and viability of implantations per dam
- mutagenicity index and dominant lethal factor were calculated by the number of dead implantations per dam (differentiation between Deciduomata and dead embryos)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
- 425 mg/kg bw: amount of substance which did not induce any death but symptoms such as ataxia and dyspnoea during determination of the acute toxicity
- 212.5 mg/kg bw: dose which was tolerated without recognizable symptoms or toxicity

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):

DETAILS OF SLIDE PREPARATION:

METHOD OF ANALYSIS:

OTHER:

Evaluation criteria:

- Conception criteria: calculated from the number of pregnant animals in relation to the number of mated animals
- Mutagenicity index: calculated from the percentage of dead implantations in relation to the total implantations
- Dominant lethal factor: frequency in per cent of the dominant lethal factors is calculated from the percentage of the viable implantations of the test group in relation to the viable implantations of the control group

Statistics:

The mean values for each test group are calculated from the values of the parameters conception rate, total implantations, viable implantations, dead implantations, mutagenicity index found for each mating interval.
- Conception rate (fertilized animals/mated animals): "Fisher's exact test (one—sided)" is applied to answer the question as to significant differences between test and control animals.
- Total implantations, viable implantations, dead implantations, mutagenicity index (= dead implantations/total implantations): "Krauth's test is used to record significant differences between test and control animals.
- The "dominant lethal factors" (dominant lethal mutations) are calculated from the viable implantations. The calculation of the frequency in per cent of the dominant lethal factors (HL%) is carried out according formula:
HL% = (1- (viable implantations / females of the test group) / (viable implantations / females of the control group)) x 100
Significant differences between test and control group were printed by a recorder. Significances at the 5% level were marked by *, significances at the 1% level by **.

Sex:

male/female

Genotoxicity:

negative

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not examined

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 0 - 1183 mg/kg bw
- Clinical signs of toxicity in test animals: After intraperitoneal administration the LD50 in male mice is 1183 mg/kg bw. Deaths occurred after administration of doses down to 620 mg/kg.

Clinical symptoms (male mice):

- 425 mg/kg bw induced apathy, ataxia, irregular breathing as well as prone and lateral position, which dissapeared after 1 h. 1 animal died one day after administration.

- 212.5 mg/kg bw was tolerated by all animals without recognizable clinical signs of toxicity and morbidity.

 

Body weight (male mice):

- the body weight development was the same in the animals treated and in those receiving the CMC solvent

 

Macroscopic examination (male mice):

- no pathological changes of the internal organs of mice of all test groups

 

Conception rate (female mice):

- during the 1st mating interval the conception rate was reduced in both desage groups (significance 99 %)

- during all other mating intervals the conception rate varied in all animals treated as well as in those of the CMC solvent control group

 

Total implantations (female mice):

- the average number of total implantations was reduced in both dosage groups during the 4th mating interval (significance 99%)

- no changes observed during all other mating intervals

 

Viable implantations (female mice):

- during the 4th matihg interval following the administration of 212.5 mg/kg (significance 95%) and 425 mg/kg (significance 99%)

- no changes observed during all other mating intervals

 

Dead implantations, mutagenicity index (female mice):

- increased percentage of dead implantations after the 5th and 9th mating interval at a dosage level of 212.5 mg/kg (significance 95%)

- percentage of dead implantations did not change after any other interval

 

Dominant lethal factor (female mice):

- the frequency of the dominant lethal factors was increased after the 4th mating interval in the 212.5 mg/kg test group (significance 95%) and in the 425.0 mg/kg test group (significance 99%)

- changes were not observed after any other interval

- the reduction of the total implantations in both dosage groups following the 4th mating interval and the resulting smaller number of viable implantations necessarily leads to significant differences in the dominant lethal factors as compared to the control group. The findings for the total and viable implantations, respectively, still range within the biological variability and cannot be related to the administration of the substance.

- after the mating intervals 5 and 9 (significance 95%) the percentage of dead (= mutagenicity index) in the low dosage group mg/kg) too, is within the biological variability. Therefore, an influence of the sbstance administered is to be excluded due to the absence of trends

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2. Nov. 1993 - 28. Oct. 1994

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:

yes

Remarks:

No positive response in positive control of Exp.1 with Cyclophosphamide. No analytical verification of substance bloodlevel

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian bone marrow chromosome aberration test

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River GmbH, WIGA, Sulzfeld,
- Age at study initiation: Not specified
- Weight at study initiation: Animals witha mean weight of about 28g were used for the study.
- Assigned to test groups randomly: yes, under following basis: according to a randomization plan prepared with an appropriate computer program.
- Fasting period before study: Not specified
- Housing: During the test animals were housed individually in Makrolon cages type MI
- Diet: Standardized pelleted feed (Kliba Haltungs Diät, Klingenthalmühle AG, Kaiseraugst, Switzerland)
- Water: ad libitum
- Acclimation period: 2 weeks in groups of 5

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Air changes (per hr): Not specified
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To: Not specified

Route of administration:

intraperitoneal

Vehicle:

Aqueous solution of 0.5% carboxy methyl cellulose

Details on exposure:

Route = intraperitoneal

Duration of treatment / exposure:

once

Post exposure period:

24h and 48h

Dose / conc.:

1 000 mg/kg bw (total dose)

Remarks:

Experiment 2
24h and 48h sacrifice interval

Dose / conc.:

1 000 mg/kg bw (total dose)

Remarks:

Experiment 1
24h and 48h sacrifice interval

Dose / conc.:

500 mg/kg bw (total dose)

Remarks:

Experiment 1
24h sacrifice interval

Dose / conc.:

250 mg/kg bw (total dose)

Remarks:

Experiment 1
24h sacrifice interval

No. of animals per sex per dose:

5 males and 5 females

Control animals:

yes, concurrent vehicle

Positive control(s):

20 mg Cyclophosphamide
0.15 mg Vincristine

Tissues and cell types examined:

Bone marrow smears

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
In a pretest for the determination of the acute intraperitoneal toxicity deaths were observed down to a dose of 1250 mg/kg bodyweight. 1000 mg/kg were survived by all animals but led to clinical signs such as apathy and abdominal position; the general state of the animals was poor. Therefore, a dose of 1000 mg/kg bodyweight was selected as the highest dose in the present cytogenetic study. 500 mg/kg and 250 mg/kg bodyweight were administered as further doses.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Sampling was performed 24 h and/ or 48 h after treatment

DETAILS OF SLIDE PREPARATION:
The bone marrow was prepared according to the method described by SCHMID, W. The two femora were prepared from the animals, and all soft tissues were removed. After cutting off the epiphyses, the bone marrow was flushed out of the diaphysis into a centrifuge tube using a cannula filled with fetal calf serum which was at 37°C (about 2 ml/femur). The suspension was mixed thoroughly with a pipette, centrifuged at 1500 rpm for 5 minutes, the supernatant was removed except for a few drops, and the precipitate was resuspended.
1 drop of this suspension was dropped onto clean microscopic slides, using a Pasteurpipette. Smears were prepared using slides with ground edges, the preparations were dried in the air and subsequently stained. The slides were stained in eosin and methylene blue solution for 5 minutes, rinsed in aqua dest. and then placed in fresh aqua dest. for 2 or 3 minutes. They were finally stained in Giemsa solution for 12 minutes. After being rinsed twice in aqua dest. and clarified in xylene, the preparations were embedded in Corbit-Balsam.


METHOD OF ANALYSIS:
In general, 1000 polychromatic erythrocytes from each of the male and female animals of every test group are evaluated and investigated for micronuclei. The normochromatic erythrocytes (= normocytes), which occur, are also scored. The following parameters are recorded:
-Number of polychromatic erythrocytes
-Number of polychromatic erythrocytes containing micronuclei The increase in the number of micronuclei in polychromatic erythrocytes of treated animals as compared with the solvent control group provides an index of a chromosome breaking (clastogenic) effect or of a spindle activity of the substance tested.
-Number of normochromatic erythrocytes
-Number of normochromatic erythrocytes containing micronuclei The number of micronuclei in normochromatic erythrocytes at the early sacrifice intervals represents the situation before test substance administration and may serve as a control value. A substance induced increase in the number of micro nuclei in normocytes may be found with an increase in the duration of the sacrifice intervals.
-Ratio of polychromatic to normochromatic erythrocytes: this ratio indicates an influence of the test substance specifically on the bone marrow. Number of small micronuclei (d < D/4) and of large micronuclei (d > D/4) (d= diameter of micronucleus, D = cell diameter) The size of micronuclei may give an indication on the possible mode of action of the test substance, i.e. aclastogenic or a spindle poison effect. Slides were coded before microscopic analysis.

OTHER:

Statistics:

The statistical evaluation of the data was carried out using the program system MUKERN (BASF AG). The number of micronuclei in polychromatic erythrocytes were analysed. A comparison of the dose group with the vehicle control was carried out using the Wilcoxon test for the hypothesis of equal medians. Here, the relative frequencies of cells with micronuclei of each animal were used. If the results of this test were significant, labels (+ for p < 0.05, ++for p < 0.01) were printed with the group means in the tables. This test was performed one sided. This analysis was done separately for each sex and combined for both sexes.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

other: No positive response with Cyclophosphamid in Exp.1

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Fenpropimorph was evaluated for possible mutagenic/genotoxic effects in vitro and in vivo. It was negative in the reverse mutation assays in bacteria (Ames test).

Point mutation in mammalian cells was assessed in an HPRT assay. In this study no mutagenic effects were observed. In the chromosome aberration test in vitro in Chinese hamster V79 cells a slight, statistically

not significant increase in the number of exchanges was observed in presence of S9 mix at the 18 h sampling interval only. As they occurred only at cytotoxic concentrations and in absence of a clear dose response relationship, the results of this study are equivocal at the most. In an earlier in vitro cytogenetics study in human lymphocytes fenpropimorph did not induce chromosome aberrations both with and without metabolic activation. Moreover, the absence of a chromosome damaging effect of fenpropimorph was confirmed by an in vivo cytogenetics study (micronucleus test).

Unscheduled DNA synthesis was assayed in primary rat hepatocytes in vitro and demonstrated the absence of a DNA damaging potential.

In two in vivo studies (mouse micronucleus test and dominant lethal assay in mice), the absence of a genotoxic potential of fenpropimorph in somatic and germ cells was confirmed.

It is concluded that fenpropimorph has no mutagenic or genotoxic potential.

Justification for classification or non-classification

The performed genotoxicity tests do not indicate a genotoxic potential of Fenpropimorph. Therefore, classification according to Regulation (EC) 1272/2008 is not warranted.