Etocrilene

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

JuIy 24, 2000 - November 6, 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes

Remarks:

BASF, Experimental Toxicology and Ecology

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Etocrilene

EC Number:

226-029-0

EC Name:

Etocrilene

Cas Number:

5232-99-5

Molecular formula:

C18H15NO2

IUPAC Name:

ethyl 2-cyano-3,3-diphenylprop-2-enoate

Details on test material:

- Physical state: Solid (powder)
- Analytical purity: > 98.5 %
- Storage condition of test material: Room temperature

Method

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (HGPRT)

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media:
1. Ham's F12 medium with glutamine and hypoxanthine supplemented with 10% (v/v) fetal calf serum (FCS). During exposure to the test substance, Ham's F12 medium was used without FCS supplementation. In case of continuous treatment Ham's F12 medium with FCS supplementation was used.
2. Pretreatment medium ("HAT medium"): FCS-supplemented Ham's F12 medium with glutamine and hypoxanthine containing Hypoxanthine (13.6 µg/ml), Aminopterin (0.18 µg/ml) and Thymidine (3.88 µg/ml).
3. Selection medium ("TG medium"): Glutamine- and FCS-supplemented, hypoxanthine-free Ham's F12 medium with 6-thioguanine at a final concentration of 10 µg/mI.
All media were supplemented with 1% (v/v) penicillin/streptomycin (10,000 IU /10,000 µg/ml) and 1 % (v/v) amphotericine B (250 µg/mI)

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver fraction (S9)

Test concentrations with justification for top dose:

1st experiment
without S-9 mix: 0; 0.313; 0.625; 1.25; 2.5; 5.0; 10.0 µg/ml
with S-9 mix (S-9 fraction : cofactors = 3 : 7): 0; 15.625; 31.25; 62.5; 125; 250; 500 µg/ml

2nd experiment
without S-9 mix: 0; 0.625; 1.25; 2.5; 5; 10; 20 µg/mI
with S-9 mix (S-9 fraction : cofactors = 1 : 9): 0; 12.5; 25; 50; 100; 200; 400 µg/mI

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation Migrated to IUCLID6: 300 µg/mI culture medium added in a volume of 1.0 ml.

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

With metabolic activation Migrated to IUCLID6: 10 µg/mI culture medium added in a volume of 1.0 ml.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

RATIONAL FOR DOSE SELECTION:
The experimental doses were determined from an appropriate pretest with cultures exposed for the duration of 4 hours to a wide dose range of the test article, i.e. 1 µg/ml - 3,000 µg/ml culture medium both without S-9 mix and after adding a metabolizing system. The cloning efficiency was strongly affected without S-9 mix from about 10 µg/ml onward. With metabolic activation the cloning efficiency was only slightly influenced from about 500 µg/ml onward but the colony size was very small demonstrating a test substance influence an colony growth. Test substance precipitation was observed from about 50 µg/ml onward. On the basis of the findings from the pretest, the following doses were selected as top doses:
- Without and with S-9 mix, 4 hours exposure time: 10 µg/ml
- With S-9 mix, 4 hours exposure time: 500 µg/ml

DURATION
- Attachment period: 24 hours
- Exposure duration: 4 hours (with and without S9 mix)
- Expression time (cells in growth medium): 7-9 days
- Selection time (if incubation with a selection agent): 1 week (in TG Medium)
- Fixation time (start of exposure up to fixation or harvest of cells): about 2 weeks

SELECTION AGENT (mutation assays): 6-thioguanine
STAIN (for cytogenetic assays): Colonies were fixed with methanol and stained with Giemsa.

NUMBER OF REPLICATIONS: 2 per dose

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency (survival of treatment and cell viability without mutant selection); mutant frequency; cell morphology after test substance exposure.

OTHER
pH values and osmolality were measured. The solubility of the test substance in the vehicle used and in the aqueous culture medium about 3 hours after treatment was checked to ensure proper culturing and to avoid extreme treatment conditions.

Evaluation criteria:

EVALUATION CRITERIA:
The criteria for a positive response are:
- Increases of the corrected mutation frequencies above the concurrent negative control values and above 15 mutants per 10^6 clonable cells and/or the evidence of a dose-response relationship
- Reproducibility of any increase in mutant frequencies.
- A statistically significant increase in mutant frequencies and the evidence of a dose-response relationship.
Isolated increases of mutant frequencies above 15 mutants per 10^6 clonable cells or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.

The test chemical is considered non-mutagenic according to the following criteria.
-The corrected mutation frequency in all dose groups is within the historical control range and is not significantly above the concurrent negative control.

Statistics:

Due to the negative findings, a statistical evaluation was not carried out.

Results and discussion

Test results

Test results 1

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The number of colonies was decreased from about 250 µg/mI onward in the 1st experiment and from about 25 µg/mI onward in the 2nd experiment. Cell density was reduced from about 250 µg/mI onward in the 1st experiment and from 50 µg/ml in the 2nd experiment

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 20 µg/ml number of colonies was decreased and ceII density was reduced.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: pH remained unaffected during the experiments
- Effects of osmolality: Osmolality remained unaffected during the experiments.
- Precipitation: Test substance precipitation was observed from about 50 µg/ml onward.

COMPARISON WITH HISTORICAL CONTROL DATA:
The mutant frequencies at any concentration were within the range of the historical control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Cell morphology: Slightly reduced attachment observed with S9 mix at 500 µg/ml

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

EXPERIMENTAL RESULT

Experiment I, 4 h exposure:

	concentration (µg/ml)	S9 Mix	absolute cloning efficiency I (%)	relative cloning efficiency I (%)	absolute cloning efficiency II (%)	relative cloning efficiency II (%)	mutant frequency / 10^6cells
DMSO		-	79.3	100	98.3	100	7.56
Positive control EMS	300	-	67.9	85.6	89.5	91	377.75
Test item	0.313	-	81.8	103.2	91.5	93.1	1.81
Test item	0.625	-	75	94.6	98.8	100.5	3.85
Test item	1.25	-	79.2	99.9	92.9	94.5	7.83
Test item	2.5	-	84	105.9	91.2	92.8	15.41
Test item	5	-	80.6	101.6	103.7	105.5	7.06
Test item	10	-	80.6	101.6	98.2	99.9	9.52
DMSO		+	91.2	100	100.4	100	1.11
Positive control MCA	10	+	95.5	95.5	91.4	91	81.11
Test item	15.625	+	89.5	98.1	95.9	95.5	6.38
Test item	31.25	+	88.7	97.3	100.6	100.2	3.76
Test item	62.5	+	83.9	92	98.9	98.5	1.73
Test item	125	+	89.4	98	96.2	95.8	11.61
Test item	250	+	32.7	35.9	96.3	95.9	8.18
Test item	500	+	0	0	98.8	98.4	10.5

Experiment II, 4 h exposure:

	concentration (µg/ml)	S9 Mix	absolute cloning efficiency I (%)	relative cloning efficiency I (%)	absolute cloning efficiency II (%)	relative cloning efficiency II (%)	mutant frequency / 10^6cells
DMSO		-	79.8	100	81.2	100	1.37
Positive control EMS	300	-	76.4	95.7	88.5	109	211.78
Test item	0.625	-	78.7	98.6	89.4	110.1	0.29
Test item	1.25	-	78.4	98.2	96.5	118.8	2.61
Test item	2.5	-	84.4	105.8	90.7	111.7	7.03
Test item	5	-	69.9	87.6	77	94.8	0.71
Test item	10	-	77.7	97.4	91.7	112.9	7.22
Test item	20	-	9.6	12	88.6	109.1	0.96
DMSO		+	80	100	90.7	100	2.17
Positive control MCA	10	+	75.5	94.4	73.6	81.1	140.51
Test item	12.5	+	58.5	73.1	83.8	92.4	0.62
Test item	25	+	23.9	29.9	95.7	105.5	0.52
Test item	50	+	15.6	19.5	80.6	88.9	7.31
Test item	100	+	8.5	10.6	91.2	100.6	0.59
Test item	200	+	6	7.5	91.7	101.1	1.73
Test item	400	+	3.4	4.3	87.2	96.1	2.41

- Mutant frequency was corrected on the basis of the absolute cloning efficiency II at the end of the expression period

- Cloning efflciency I (survival): Determined for each test group after the exposure period in parallel cultures.

- Cloning efficiency II (viability): Determined for each test group at the end of the expression period in parallel cultures.

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions of this assay, the test substance has no mutagenic activity in vitro in the CHO/HPRT forward mutation assay.

Executive summary:

In a GLP compliant study following OECD guideline no. 476, the test article was tested for its ability to induce gene mutations at the

hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Two independent experiments were carried out, both with and without the addition of Aroclor-induced rat liver S-9 mix (exogenous metabolic activation).

According to an initial range-finding cytotoxicity test for the determination of the experimental doses and taking into account the cytotoxicity actually found in the main experiment, the following doses were evaluated in the 1st experiment after an exposure period of 4 hours:

without S-9 mix: 0; 0.313; 0.625; 1.25; 2.5; 5.0; 10.0 µg/ml

with S-9 mix (S-9 fraction cofactors = 3 7): 0; 15.625; 31.25; 62.5; 125; 250; 500 µg/ml

In a 2nd experiment for confirmation of the results of the 1st experiment, the following doses were evaluated after an exposure period of 4 hours with S-9 mix and without metabolic activation:

without S-9 mix: 0; 0.625; 1.25; 2.5; 5; 10; 20 µg/mI

with S-9 mix (S-9 fraction cofactors = 1: 9): 0; 12.5; 25; 50; 100; 200; 400 µg/mI

After an attachment period of 20 - 24 hours and a treatment period of 4 hours both with and without metabolic activation, an expression phase of about 7 - 9 days and a selection period of about 1 week followed. The colonies of each test group were fixed with methanol, stained with Giemsa and counted. The negative controls (vehicle controls) gave mutant frequencies within the range

expected for the CHO cell line. Both of the positive control chemicals, i.e. EMS and MCA, led to the expected increase in the frequencies of forward mutations. On the basis from the results of the present study, the test substance did not cause any increase in the mutant frequencies either without S-9 mix or after adding a metabolizing system in two experiments performed independently of each other. Thus, under the experimental conditions of this assay, the test substance has no mutagenic activity in vitro in the CHO/HPRT forward mutation assay.