2-methylbenzene-1,4-diyl bis{4-[3-(acryloyloxy)propoxy]benzoate}

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 AUG 2005 - 14 JUL 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

This study was conducted according to Good Laboratory Practice (GLP) and followed the OECD Guideline for the testing of Chemicals: Genetic Toxicology: 473 In vitro mammalian chromosome aberration test.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

21 Jul 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

425-560-7

EC Name:

-

Cas Number:

174063-87-7

Molecular formula:

C33H32O10

IUPAC Name:

2-methylbenzene-1,4-diyl bis{4-[3-(acryloyloxy)propoxy]benzoate}

Test material form:

solid: crystalline

Method

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CHO cells, supplied by Dr S Galloway, West Point, PA, USA, are maintained at Covance Laboratories Limited in tissue culture flasks containing McCoy's 5A medium including 10% (v/v) foetal calf serum (FCS), and 100 µg/mL gentamycin. They are subcultured regularly at low density, and before overgrowth occurs, to maintain low aberration frequencies. Stocks of cells preserved in liquid nitrogen are reconstituted for each experiment so as to maintain karyotypic stability. The cells are screened for mycoplasma contamination.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 after induction using Aroclor 1254
Type and composition of metabolic activation system:
- source of S9 : male Sprague Dawley rats induced with Aroclor 1254
- method of preparation of S9 mix : The batches of MolTox S-9 were stored frozen in aliquots at -80°C and thawed just prior to use. Glucose-6-phosphate (180 mg/mL), NADP (25 mg/mL), 150 mM KCl and rat liver S-9 were mixed in the ratio 1:1:1:2. An aliquot of the resulting S-9 mix was added to each cell culture designated for treatment in the presence of S-9 to achieve the required final concentration in a total of 10 mL. The final concentration of liver homogenate in the test system was 2%. Cultures treated in the absence of S-9 received an equal volume of 150 mM KCl.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Each batch was checked by the manufacturer for sterility, protein content, ability to convert known promutagens to bacterial mutagens and cytochrome P-450-catalyzed enzyme activities (alkoxyresorufin-Odealkylase activities).

Test concentrations with justification for top dose:

Top concentration in experiment was determined based on range-finding test

concentrations used for cell analysis:
Experiment I:
200.0, 300.0, 350.0 µg/mL (-S9)
200.0, 250.0 , 300.0 µg/mL (+S9)
The highest concentrations chosen for analysis (3 treatment + 17 recovery hours), 350.0 µg/mL in the absence of S-9 and 300.0 µg/mL in the presence of S-9, induced approximately 50% and 42% reduction in population doubling, respectively.

Experiment II
5, 100, 1500, 3.355 µg/mL (-S9)
50, 250, 300 µg/mL (+S9)
The highest concentrations chosen for analysis (treatment + recovery hours), 1500 µg/mL (20+0) and 3.355 µg/mL (44+0) in the absence of S-9 and 300.0 µg/mL (3+17 and 3+41) in the presence of S-9, induced approximately 39%, 47%, 59% and 25% reduction in population doubling, respectively.

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Sterile DMSO was added to cultures designated as negative controls

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

cyclophosphamide

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : duplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1.929 x 10E6 (20+0, -S9, 3+17, +S9), 1.097 x 10E6 (44+0, -S9, 3+41, +S9)
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment:
2 continuous treatments (treatment+recovery time +/-S9: 20+0 -S9; 44+0 -S9): Treatment media remained on cultures receiving the continuous treatment until sampling, that is, 20 or 44 hours after the beginning of treatment.
3 pulse treatments (treatment+recovery time +/-S9: 3+17 +S9; 3+17 - S9; 3+41 +S9): Cultures received pulse treatments (both in the absence and presence of S-9) for 3 hours only. They were then washed twice with sterile saline, and fresh medium containing foetal calf serum and gentamycin added. Cultures were incubated for a further 17 or 41 hours before harvesting.
- Harvest time after the end of treatment (sampling/recovery times):
2 continuous treatments (treatment+recovery time +/-S9: 20+0 -S9; 44+0 -S9): 0 h
3 pulse treatments (treatment+recovery time +/-S9: 3+17 +S9; 3+17 - S9; 3+41 +S9): 17 h , 41 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: Colchicine (1 µg/mL) for 2 h
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): Cells were resuspended in 4mL pre-warmed hypotonic (0.075 M) KCl and incubated at 37°C for 5 minutes to allow cell swelling to occur. Cells were then fixed by dropping the KCl suspension into an equal volume of fresh, ice-cold methanol/glacial acetic acid (3:1, v/v). The fixative was changed by centrifugation (approximately 200 x g for 5 minutes) and resuspension. This procedure was repeated twice (centrifuging at approximately 1250 x 'g', 2-3 minutes) until the cell pellets were clean.
Cells were kept in fixative in the refrigerator before slides were prepared but slides were not made on the day of harvest to ensure cells were adequately fixed. Cells were pelleted and resuspended in a minimal amount of fresh fixative (if required) so as to give a milky suspension. Several drops of 45% (v/v) aqueous acetic acid were added to each suspension to enhance chromosome spreading, and several drops of suspension were transferred to clean microscope slides. After the slides had dried on a warm plate the cells were stained for 5 minutes in 4% (v/v) filtered Giemsa stain in Gurr's pH 6.8 buffer. The slides were rinsed, dried and mounted with coverslips.
- Number of cells spread analysed per concentration (number of replicate cultures and total number of cells scored): Where possible, one hundred metaphases from each treatment/control were analysed for chromosome aberrations.
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): Only cells with 19-23 chromosomes were considered acceptable for analysis of structural aberrations. Classification of structural aberrations was based on the scheme described by ISCN (ISCN (1995) An International System for Human Cytogenetic Nomenclature. Editor Felix Mitelman; S Karger, Switzerland.). Under this scheme, a gap is defined as a discontinuity less than the width of the chromatid and no evidence of displacement of the fragment and a deletion is defined as a discontinuity greater than the width of the chromatid and/or evidence of displacement of the fragment
- Determination of polyploidy, endoreplication: Any cell with more than 23 chromosomes, that is polyploid, endoreduplicated and hyperdiploid cells, observed during this search was noted and recorded separately.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative population doubling (RPD), mitotic index (MI)

Evaluation criteria:

A test article is considered as positive in this assay if:
1. the proportions of cells with structural aberrations at one or more concentrations exceeds the normal range in both replicate cultures, and
2. a statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) occurs at these doses.
3. a concentration-related trend in the proportion of cells with structural aberrations (excluding gaps).
A test article is considered positive in this assay if all of the above criteria are met.
A test article is considered negative in this assay if none of the above criteria are met.

Statistics:

Descriptive statistics using Fisher's exact test

Richardson C, Williams D A, Allen J A, Amphlett G, Chanter D 4 and Phillips B (1989) Analysis of data from in vitro cytogenetic assays. In "Statistical Evaluation of Mutagenicity Test Data", (UKEMS Guidelines Sub-committee Report, Part III), Ed D J Kirkland, Cambridge University Press, pp 141-154.

Results and discussion

Test results

Test results 1

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH, osmolality: Measurements on post-treatment media from the range-finder in the absence and presence of S-9 indicated that the test article had no marked effect on osmolality (greater than a shift of 50 mOsm/kg) or pH (shift of greater than 1 pH unit) as compared to concurrent vehicle controls
- Precipitation and time of the determination: It was noted that precipitate was visible at the time of harvest at a concentration of 350.0 pg/mL. As such, the test article was present during the recovery stage of treatment.
- Definition of acceptable cells for analysis: Only cells with 19-23 chromosomes were considered acceptable for analysis of structural aberrations. Any cell with more than 23 chromosomes, that is polyploid, endoreduplicated and hyperdiploid cells, observed during this search was noted and recorded separately.

RANGE-FINDING/SCREENING STUDIES (if applicable): It should be noted that two separate range-finder treatments were performed due to limited culture availability. In the first range-finder, cultures were treated at 3+17 hours, - and +S-9 and 20+0 hours -S-9. In the second, cultures wisre treated at 44+0 hours -S-9 and 3+41 hours +S-9.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : The proportion of cells with structural aberrations (excluding gaps) in negative control cultures fell within the normal range. The positive control chemicals NQO and CPA induced statistically significant increases in the number of cells with structural aberrations.

For all test methods and criteria for data analysis and interpretation:
please refer to result tables attched in 'Attached background material'

Chromosome aberration test (CA) in mammalian cells:
- please refer to result tables attched in 'Attached background material'
- Genotoxicity results (for both cell lines and lymphocytes)
o Definition for chromosome aberrations, including gaps : Classification of structural aberrations was based on the scheme described by ISCN (ISCN (1995) An International System for Human Cytogenetic Nomenclature. Editor Felix Mitelman; S Karger, Switzerland.). Under this scheme, a gap is defined as a discontinuity less than the width of the chromatid and no evidence of displacement of the fragment and a deletion is defined as a discontinuity greater than the width of the chromatid and/or evidence of displacement of the fragment.
o Number of cells scored for each culture and concentration, number of cells with chromosomal aberrations and type given separately for each treated and control culture, including and excludling gaps : At least 160 cells out of an intended 200 were analysable at each dose level, unless 10 or more cells showed structural aberrations other than gaps, observed during analysis.
please refer to result tables attched in 'Attached background material'
o Changes in ploidy (polyploidy cells and cells with endoreduplicated chromosomes) if seen : A single culture at the highest concentration analysed (3.355 pg/mL) following 44+0 hour -S-9 treatment exhibited an increase in numbers of polyploid cells, but this was not reproduced between replicate cultures.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Negative (solvent/vehicle) historical control data:
-S9: 25 studies, 113 cultures
range 0-8, mean 1.5, SD 1.74 (structural aberrations including gaps); range 0-7, mean 1.01, SD 1.37 (structural aberrations excluding gaps)
+S9: 17 studies, 64 cultures
range 0-6, mean 1.66, SD 1.45 (structural aberrations including gaps); range 0-5, mean 1.25, SD 1.23 (structural aberrations excluding gaps)

Applicant's summary and conclusion

Conclusions:

It is concluded that under the experimental conditions employed, the test material induced both structural and numerical chromosome aberrations in cultured Chinese hamster ovary (CHO) cells following pulse 3+17 hour treatment in the presence of metabolic activation (S9). No such increases in structural or numerical aberrations were observed following 3+41 hour +S9 treatment at an equivalent concentration, nor following treatments in the absence of S9 (3+17, 20+0 and 44+0 hour treatments) at concentrations either up to the limit of cytotoxicity, or, limited by solubility, in the test system.

Executive summary:

The registered substance was tested for cytogenicity according to OECD Guideline 473 following GLP.

Purpose
The purpose of the in vitro chromosome aberration test is to identify agents that cause structural chromosome aberrations in cultured mammalian cells thus providing information on possible health hazards for the test material and serve as a rational basis for risk assessment to the genotoxic potential of the test item in human.

Study Design
The test material was tested in an in vitro cytogenetics assay using duplicate cultures of Chinese hamster ovary (CHO) cells in two independent experiments. Treatments covering a broad range of doses, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S9). The test article was dissolved in sterile anhydrous analytical grade dimethyl sulphoxide (DMSO) and the highest dose level used in the main experiments, 1500 µg/mL, was determined following a preliminary cytotoxicity range-finding experiment.

Results
Cytotoxicity based on PD
In Experiment 1, treatment in the absence and presence of S9 was for 3 hours followed by a 17-hour recovery period prior to harvest (3+17). The S9 mix used was prepared from a rat liver post-mitochondrial fraction from Aroclor 1254 induced animals. The test article dose levels for chromosome analysis were selected by evaluating the effect of the test item on population doubling. Chromosome aberrations were analysed at three dose levels; the highest concentrations chosen for analysis, 350.0 µg/mL in the absence of S9 and 300.0 µg/mL in the presence of S9, induced approximately 50% and 42% reduction in population doubling, respectively.

In Experiment 2, treatment in the absence of S9 was continuous for either 20 hours (20+0) or 44 hours (44+0). Treatment in the presence of S9 was either for 3 hours only followed by a 17-hour recovery period prior to harvest (3+17), or for 3 hours followed by a 41-hour recovery period prior to harvest (3+41). Chromosome aberrations were analysed at up to three dose levels and the highest concentrations chosen for analysis, 1500 µg/mL (20+0) and 3.355 µg/mL (44+0) in the absence of S9 and 300.0 µg/mL (3+17 and 3+41) in the presence of S9, induced approximately 39%, 47%, 59% and 25% reduction in population doubling, respectively.

Appropriate negative (solvent) control cultures were included in the test system in both experiments under each treatment condition. The proportion of cells with structural aberrations in these cultures fell within historical solvent control ranges. 4-Nitroquinoline 1-oxide (NQO) and cyclophosphamide (CPA) were employed as positive control chemicals in the absence and presence of liver S9, respectively. Cells receiving these were sampled in each experiment, 20 hours after the start of treatment; both compounds induced statistically significant increases in the proportion of cells with structural aberrations. Positive controls were included with both treatments in Experiment 1, but only with the 20+0 hour -S9 and 3+17 +S9 treatments in Experiment 2.

Structural Chromosome aberration analysis
Treatment of cultures with test substance in the absence of S9 in both experiments resulted in frequencies of cells with structural aberrations that were similar to those observed in concurrent negative controls for the majority of concentrations analysed. Numbers of aberrant cells (excluding gaps) for the majority of  treated cultures fell within historical negative control (normal) ranges.

Pulse 3+17 hour treatment of cultures with the test substance in the presence of S9 resulted in frequencies of cells with structural aberrations that were significantly higher (p≤0.05) than those observed in concurrent vehicle controls for the highest two concentrations analysed (250 and 300 µg/mL) in both treatments. These increases were observed to be dose-related. Large increases in cells with structural aberrations, exceeding historical vehicle control values were observed in both replicate cultures at the concentration of 300 µg/mL in both treatments. Single cultures at 200 and also at 250 µg/mL in Experiment I also exhibited aberrant cell frequencies exceeding historical vehicle control values, although such increases were not observed in the replicate cultures at either concentration. All other treated cultures exhibited numbers of aberrant cells that fell within historical vehicle control (normal) values. The high concentration analysed (300 µg/mL) induced 42% and 59% cytotoxicity (based on population doublings [PD]) in Experiments 1 and 2, respectively.

Pulse 3+41 hour treatment of cultures with the test item in the presence of S9 (Experiment 2) resulted in frequencies of cells with structural aberrations that were similar to those observed in concurrent vehicle controls at a concentration of 300 µg/mL (a concentration that induced 25% cytotoxicity based on PD). The aberrant cell frequency of both replicate cultures at this concentration fell within normal values.

Numerical Chromosome aberration analysis
Frequencies of cells with numerical aberrations fell within the historical negative control (normal) range for the majority of cultures treated in the absence of S9 (both experiments).

Pulse 3+17 hour treatments in the presence of S9 resulted in large increases in numerical aberrations for the majority of concentrations analysed (both experiments). These increases were attributable to endoreduplicated cells and clearly exceeded historical vehicle control ranges for the majority of treated cultures. Dose related increased toxicity was observed in both treatments over the concentration range analysed (200-300 µg/mL and 50-300 µg/mL, Experiments 1 and 2 respectively).

No such increases in numerical aberrations were observed following 3+41 hour +S9 treatment at a concentration of 300 µg/mL.

Conclusion
It is concluded that under the experimental conditions employed, the test material induced both structural and numerical chromosome aberrations in cultured Chinese hamster ovary (CHO) cells following pulse 3+17 hour treatment in the presence of metabolic activation (S9). No such increases in structural or numerical aberrations were observed following 3+41 hour +S9 treatment at an equivalent concentration, nor following treatments in the absence of S9 (3+17, 20+0 and 44+0 hour treatments) at concentrations either up to the limit of cytotoxicity, or, limited by solubility, in the test system.

It should be noted that this particular assay is primarily designed to detect structural rather than numerical chromosome aberrations and the biological relevance of endoreduplication in vitro is questionable.