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

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

266-719-9

EC Name:

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

Cas Number:

67564-91-4

Molecular formula:

C20H33NO

IUPAC Name:

(2R,6S)-4-[3-(4-tert-butylphenyl)-2-methylpropyl]-2,6-dimethylmorpholine

Method

Target gene:

HPRT

Species / strain

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

Controls

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.

Results and discussion

Test results

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

Applicant's summary and conclusion

Conclusions:

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