Benzoic acid, 4-[[[4-[(1-oxo-2-propenyl)oxy]butoxy]carbonyl]oxy]-, 2-methyl-1,4-phenylene ester

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

- Physical state: powder / grey-white
- Analytical purity: 95.8% (per weight; HPLC)
- Lot/batch No.: ZD 1151/ 31 (production: 03-Dec-1997)
- Storage condition of test material: refrigerator, protected from light.

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

supernatant of rat liver homogenates (5 male SD rats receive a single intrapentoneal injection of 500 mg Aroclor 1254 [as a solution in com oil with a concentration of 200 mg/ml] per kg body weight 5 days before sacrifice) mixed with cofactors

Test concentrations with justification for top dose:

Pretests for dose selection: 50 - 5000 µg/ml; first experiment: 1, 10, 100, 500 and 1000 µg/ml; second experiment: 1, 5, 10, 50 and 100 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: due to the limited solubility of the test substance in water, DMSO was selected as the vehicle, which had been demonstrated to be suitable in the V79 in vitro cytogenetic test and for which historical control data are available.

Controls

Untreated negative controls:

other: historical control data exist, demonstrating that no deleterious or mutagenic effects are induced by the chosen solvent

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: - without metabolic activation: 350 pg ethyl methanesulfonate (EMS)/mI culture medium added in a volume of 1.0 ml; - with metabolic activation: 0.5 pg cyclophosphamide (CPP)/mI culture medium added in a volume of 1.0 ml.

Remarks:

The stability of EMS and CPP is well-defined under the selected culture conditions, since both positive control articles are weII-established reference clastogens.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 18 or 28 hours
- Fixation time (start of exposure up to fixation or harvest of cells):

SPINDLE INHIBITOR (cytogenetic assays): 2 - 3 hours prior to harvesting the cells, 0.2 pg colcemid/mI culture medium (1 pg colcemid dissolved in 0.1 ml PBS/culture) was added to each chamber in order to arrest mitosis in the metaphase.
STAIN (for cytogenetic assays): 5 ml of fixative (methanol glacial acetic acial/3: 1, after hypotonic treatment using 5 ml of a 0.4% KCI solution) was added to the cells, kept for at least 15 minutes and then replaced. After about another 10 minutes, the fixative was replaced again and kept for at least 5 minutes at room temperature for complete fixation. The preparations were dried in the air and subsequently stained in a solution of Giemsa and Titrisol for 10 minutes. After being rinsed twice in purified water and clarified in xylene, the preparations were mounted using Corbit-Balsam.

NUMBER OF REPLICATIONS: the expression time was set to cover 1-1.5 cell cycle or any delayed cell cycle

NUMBER OF CELLS EVALUATED: counted for all test groups, and if cells had 20 - 22 chromosomes, they were analyzed for structural chromosome aberrations. Numerical chromosome aberrations were also recorded. Structural chromosome aberrations and numerical chromosome aberrations (so-called heteroploldies, including aneuploidy and polyploidy) were then analyzed.


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: a mitotic index based on 1000 cells/culture was determined for all test groups in both experiments. For the determination of cytotoxicity, additional cell cultures (using 25 cm 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 with the concurrent vehicle control at the end of the culture period using a counting chamber.

OTHER: About 3 hours after test substance treatment, cultures of all test groups were checked for ceII morphology, which is an indication of attachment of the cells to the slides.

Evaluation criteria:

The test chemical is to be considered positive in this assay if the following criteria are met:
- A dose-related and reproducible significant increase in the number of structural chromosomal aberrations.
- The proportion of aberrations exceeded both the concurrent negative control range and the negative historical control range.
A test substance is generally considered nonclastogenic in this test system if:
- There was no significant increase in the number of chromosomally damaged cells at any dose above concurrent control frequencies.
- The aberration frequencies were within the historical control range.

Statistics:

- The statistical evaluation of the data was carried out using the MUCHAN program System.
- The proportion of metaphases with aberrations was calculated for each group.
- 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 Bonferroni-Holm corrected versus the dose groups separately for each time and was performed one-sided.

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

40% cell count (relative to vehicle control) was observed at 500 µg/ml (without S-9 mix) during the first main experiment.

Vehicle controls validity:

valid

Untreated negative controls validity:

other: historical control data exist, demonstrating that no deleterious or mutagenic effects are induced by the chosen solvent

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS

- Precipitation: precipitation was generally observed from 10 µg/ml upwards

- Other confounding effects: according to the results of the present in vitro cytogenetic study, under most of the experimental conditions the test substance led to a slight increase in the number of structural chromosomal aberrations including and excluding gaps both without S-9 mix and after the addition of a metabolizing System in two experiments performed independently of each other selecting different exposure times (4 hours treatment and continuous treatment) and harvest times (18 and 28 hours).
These slightly elevated figures were always observed only at the selected top doses at which evident test substance precipitation was found. These precipitated test substance particles may be phagocytized leading to high local concentrations within the cells with the possible result of lysosomal damage and a subsequent release of nucleases representing an overload phenomenon. Therefore, it is highly probable that the effect observed at the highest dose levels is the result of an indirect mechanism (chromosome damage by liberated nucleases) due to treatment conditions rather than a DNA-damaging activity of the test substance.
No increase in the number of cells containing numerical chromosomal aberrations was demonstrated.
The increase in the frequencies of chromosomal aberrations induced by the positive control agents EMS and CPP clearly demonstrated the sensitivity of the test method and of the metabolic activity of the S-9 mix employed.

RANGE-FINDING/SCREENING STUDIES: the cell count was 77.3, 85.0, 69.6, 59.2, 71.5 and 61.5, respectively at 50, 100, 500 1000, 2500 and 5000 µg/ml during the pretest experiments (with precipitation generally observed from 50 µg/ml upwards; cell attachment and mitotic index not changed). Therefore, dose levels ranging from 1 to 1000 µg/ml were selected for the first and second main experiments.

ADDITIONAL INFORMATION ON CYTOTOXICITY: no cytotoxicity was observed in the second experiment (cell survival ranging from 80.8 to 121.8). Cell morphology (fibroblast-like) and attachment were not affected, in the pretest and in the 2 main experiments. Osmolality (range: 395 - 418) and pH (range: 7.5 - 7.7) were also not affected by the treatment.

Any other information on results incl. tables

1) Chromosome analysis

- First experiment

	Aberrations (%)			Exposure	Harvest
Treatment (µg/ml)	Metaphases including gaps	Metaphases including gaps	S-9 mix	time (hours)	time (hours)
DMSO	9 (4.5%)	6 (3.0%)	-	4	18
	4 (2.0%)	2 (1.0%)	+	4	18
Test substance (10)	4 (2.0%)	2 (1.0%)	-	4	18
	10 (5.0%	7 (3.5%)	+	4	18
Test substance (100)	13 (6.5%)	11 (5.5%)	-	4	18
	20 (10.0%)	8 (4.0%)	+	4	18
Test substance (500)	13 (6.5%)	13 (6.5%)	-	4	18
(from pretest)	28 (10.0%)	18 (9.0%)	-	4	18
	36 (18.0%)	13 (6.5%)	+	4	18
Positive control (350)	(20.0%)	(17.0%)	-	4	18
Positive control (0.5)	(26.0%)	(21.0%)	+	4	18

- Second experiment

	Aberrations (%)			Exposure	Harvest
Treatment (µg/ml)	Metaphases including gaps	Metaphases including gaps	S-9 mix	time (hours)	time (hours)
DMSO	17 (8.5%)	4 (2.0%)	-	4	18
	17 (8.5%)	9 (4.5%)	-	18	28
	7 (3.5%)	6 (3.0%)	+	4	28
Test substance (5)	11 (5.5%	6 (3.0%)	-	4	18
Test substance (10)	21 (10.5%)	11 (5.5%)	-	4	18
	6 (3.0%)	3 (1.5%)	+	4	28
Test substance (50)	28 (14.0%)	14 (7.0%)	-	4	18
	9 (4.5%)	6 (3.0%)	+	4	28
Test substance (100)	35 (17.5%)	13 (6.5%)	-	18	28
	7 (3.5%)	2 (1.0%)	+	4	28
Positive control (350)	(27.0%)	(22.0%)	+	4	18