2-methyl-1,4-phenylene-bis[4-(6-acryloyloxyhexyloxy)benzoate]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item was not mutagenic in bacteria with and without addition of S9 mix in a GLP conform study according to OECD Guideline 471.

The test item did not induce strutural or numerical chromosome aberrations in mammalian cells according to OECD Guideline 473.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 10 to May 18, 1995

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:

August 1983

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Version / remarks:

August 1983

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

December 1992

Deviations:

no

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 TA 102

Metabolic activation:

with and without

Metabolic activation system:

Liver S9-Mix from Aroclor 1254-pretreated rats

Test concentrations with justification for top dose:

First experiment: 0.5, 1.58, 5, 15.8, 50, 158, and 500 µg/plate
Second experiment: 5, 15.8, 50, 158, and 500 µg/plate

The highest concentration of 500 µg/plate was chosen as higher concentrations were not soluble in the solvent.

Vehicle / solvent:

- Vehicle/solvent used: acetone
- Justification for choice of solvent/vehicle: Historical data of the laboratory and experience of other research groups (Maron et al. 1981) showed that such amounts of the selected solvents have no influence on the number of spontaneous revertants of any strain.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

cumene hydroperoxide

other: 2-Aminoanthracene, Daunomycin

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 - 72 hours
-
NUMBER OF REPLICATIONS: three

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants, clearing of background lawn of non-revertant bacteria

Evaluation criteria:

A test material is defined as non-mutagenic in this assay if
- "no" or "weak increases" occur in the first and second series of the main experiment.
("Weak increases" randomly occur due to experimental variation.)

A test material is defined as mutagenic in this assay if
- a dose-related (over at least two test material concentrations) increase in the number of revertants is induced, the maximal effect is a "clear increase", and the effects are reproduced at similar concentration levels in the same test system;

- "clear increases" occur at least at one test material concentration, higher concentrations show streng precipitation or cytotoxicity, and the effects are reproduced at the same concentration level in the same test system.

In all further cases should a third test series with the bacterial strain in question be performed. lf the criteria for a positive test result are not fulfilled in at least two out of the three series, the test material is defined as being non-mutagenic in this test system.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation occurred at the highest contration tested 500 µg/plate.

RANGE-FINDING/SCREENING STUDIES: The test material was investigated in the first experimental series at seven concentrations, separated by half-log intervals, ranging from 0.5 to 500 μg per plate. Higher concentrations were not soluble in the solvent used. In the second experimental series, usually 5 concentrations including at least 4 non-toxic concentrations should be tested.

Conclusions:

With and without addition of S9-Mix as the external metabolizing system, the test item was not mutagenic under the experimental conditions described.

Executive summary:

The investigations for mutagenic potential were performed using Salmonella typhimurium TA 98, TA 100, TA 102, TA 1535, and TA 1537 as tester strains. The plate incorporation test with and without addition of liver S9-Mix from Aroclor 1254- pretreated rats was used.

The test material was tested in two series of experiments at concentrations ranging from 0.5 to 500 μg/plate. Higher concentrations were not soluble in the solvent used. Precipitation of the test material on the agar plates was macroscopically visible at the highest concentration used (500 μg/plate). Toxicity to the bacteria was not observed.

Daunomycin, N-ethyl-N'-nitro-N-nitrosoguanidine, 9-aminoacridine and cumene hydroperoxide served as strain specific positive control compounds in the absence of S9-Mix. 2-Aminoanthracene and benzo[a]pyrene were used for testing the bacteria and the activity of the S9-Mix. Each treatment with the substances used as positive controls led to a clear increase in revertant colonies, thus showing the expected reversion properties of all strains and good metabolic activity of the S9-Mix used. With and without addition of S9-Mix as the external metabolizing system, the test material was not mutagenic under the experimental conditions described.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 03, 2006 - September 20, 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

8 June 2000

Deviations:

no

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
- Source of cells: Hoffmann-La Rache, Pharma, Basel
- Suitability of cells: This cell line has a high proliferation rate and cloning efficiency. The cells have a stable karyotype and an aberration rate of about 0-5% of the metaphases.
- Methods for maintenance in cell culture if applicable: Large stocks of this clone are stored in liquid nitro gen allowing the repeated use ofthe same cell culture batch in different experiments.
- Modal number of chromosomes: 22 +/- 1
- Normal (negative control) cell cycle time: 23 to 28 hours corresponds to 1.5 normal cell cycle length

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: The cells were cultured in supplemented Dulbecco's Minima! Essential Medium (DMEM). The DMEM was supplemented with L-glutamine (4 mM), sodium bicarbonate (0.375 %), antibiotics (neomycine 0.015 %), and 10 % fetal calf serum (FCS). All incubations were performed at +37°C in a 4-5 % carbon dioxide atmosphere (100 % humidity).
- Periodically checked for Mycoplasma contamination: yes
- Other: The cells were cloned to minimize the frequency of mutants, and to stabilize the karyotype. The cells have a spontaneous aberration rate of about 0-5 % aberrant metaphases.

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 mix induced by Aroclor 1254

Test concentrations with justification for top dose:

5.0, 15.8, and 50.0 µg/mL (+/- S9 mix)
The highest concentration of the test item was chosen based on the limited solubility of the test item.

Vehicle / solvent:

- Vehicle/solvent used: acetone
- Justification for choice of solvent/vehicle: Analysis of the historical data of our laboratory and experience of other research groups showed that such amounts of the selected solvents have no influence on the mutation frequency in this test system.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

other: Griseofulvin

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding: 100.000 cells

DURATION
- Exposure duration: 5, 25, and 35 hours (- S9 mix); 5 hours (+ S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 25 and 35 hours, respectively

SPINDLE INHIBITOR (cytogenetic assays): colchicine

STAIN (for cytogenetic assays): Cells were stained in aceto-orcein, immersed in xylene and made permanent with Entellan

NUMBER OF REPLICATIONS: 2 (positive and test material); 4 (solvent control)

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The medium is replaced by KCl-solution (0.4 %) for hypotonic treatment. For fixation of cells fixative ( methanol: acetic acid, 3: 1) is added and then renewed 2 times. The slides are then stained in aceto-orcein, immersed in xylene and made permanent with Entellan®.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: 100

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index, cell viability

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes

The frequnecy determinaton of polyploid cells and endoreduplications (chromosomes with 4, 8, 16 ... chromatids) is based on scoring 1000 mitoses per slide, and estimation of the mitotic index on scoring 1000 cells per slide.

Evaluation criteria:

A test material is defined as being unambiguously negative or non-clastogenic in this test system if no statistically significant increase in the number of aberrant metaphases per 100 cells, as compared to the actual negative control, occurs at any test material concentration.
A test material is positive or clastogenic in this test system if
- a statistically significant, dose-related increase in the number of aberrant metaphases per 100 cells occurs or
- a statistically significant increase in the number of aberrant metaphases per 100 cells is reproduced at the same test material concentration in independent experiments.
In both cases, however, the number of aberrant metaphases should be above the range defined as the historical negative controls of the laboratory and the biological relevance of the results has to be discussed.

In all other cases, further decisions for testing strategies should be made following the scientific evaluation of all existing data including those of nontoxicological investigations.

Statistics:

Descriptive statistics
For all groups, mean values were calculated for the following parameters:
• the percentage of observed aberrant metaphases/culture (gaps included),
• the percentage of observed aberrant metaphases/culture (gaps excluded),
• the number of mitoses per 1000 cells/ culture
• the number of polyploid cells per 1000 mitotic cells/culture

Statistical tests
For further statistical analysis the numbers of aberrant metaphases (gaps excluded) were used. Pairwise comparisons within each experimental series. Each treatment group was compared with the negative control. For the comparisons the Exact Fisher Test was performed against one-sided alternatives. The p-values of these comparisons are presented in the tables section.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Precipitation: at the highest concentration tested, i.e. 50 µg/mL

RANGE-FINDING/SCREENING STUDIES: Cytotoxicity of the test item was examined in the range of 0.05 - 50.0 g/mL with and without S9 mix. No clear reduction of mitotic index or cell viability (MTT-test) were induced by the test material. However, the highest concentration tested was in excess of the solubility limit in the culture medium. Thus, this was considered a suitable maximum concentration.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
-S9 mix (EMS): mean: 13.3 +/- 7.9; range: 4.5 - 5.5
+S9 mix (CPA): mean: 11.7 +/- 5.2; range: 4.0 - 28.5
- Negative (solvent/vehicle) historical control data:
-S9 mix: mean: 1.4 +/- 1.0; range: 0 - 5.0
+S9 mix: mean: 1.6 +/- 1.0; range 0 - 5.0

- Other: Treatment of cultures with the test item in the absence and presence of S9 mix resulted in frequencies of cells with polyploidic metaphases that were very similar or identical to those seen in the concurrent negative controls. No endoreduplications were observed.

Conclusions:

In conclusion, treatment of V79 cell cultures with the test item, did not increase the proportion of cells with aberrant chromosomes.The test item was not clastogenic in this in vitro test system.

Executive summary:

The test item was investigated in two experimental series for induction of chromosomal aberrations in V79 Chinese hamster cells in vitro according to OECD Guideline 473. This also included examinations on whether or not the test material may have the potential to induce numerical aberrations, i. e. an increase in number of polyploid cells.

Due to the solubility of the test item, the concentration ranging from 5.0, 15.8, and 50.0 µg/mL with and without S9 mix. In the absence of S9 mix, cells were exposed to the test item for 5, 25 or 35 hours, respectively. In the presence of S9 mix, cells were treated for 5 hours with the test item.

The positive control test materials, EMS and CPA, induced the expected clear increase in the proportion of cells with chromosomal aberrations. Griseofulvin induced a clear increase in the number of polyploid cells.

The test item precipitated in the culture medium at the highest concentration tested, i.e. 50.0 μg/mL. No clear cytotoxic effects, i.e. reduction in mitotic index or cell viability (MTT test), were induced by the test material.

The mean values of aberrant metaphases (gaps excluded) in the negative controls of both experiments (- and + S9 mix) ranged from 0.5 to 1.75 %.

The test item did not show any biologically nor statistically relevant increase in the number of aberrant metaphases. Furthermore, no treatment related increase of endoreduplications or polyploid cells was observed, i.e. neither structural nor numerical aberrations were detected.

In conclusion, treatment of V79 cell cultures with the test item, did not increase the proportion of cells with aberrant chromosomes. The test item was not clastogenic in this in vitro test system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames test

The investigations for mutagenic potential were performed using Salmonella typhimurium TA 98, TA 100, TA 102, TA 1535, and TA 1537 as tester strains with and without S9 Mix in a GLP conform study according to OECD Guideline 471. The test material was tested in two series of experiments at concentrations ranging from 0.5 to 500 μg/plate. Higher concentrations were not soluble in the solvent used. Toxicity to bacteria was not observed. While the positive controls showed a clear increase in revertant colonies, the test material was not mutagenic with and without S9 Mix under the experimental conditions described.

In vitro chromosome aberration test

The test item was investigated in two experimental series for induction of chromosomal aberrations in V79 Chinese hamster cells in vitro according to OECD Guideline 473. This also included examinations on whether or not the test material may have the potential to induce numerical aberrations, i. e. an increase in number of polyploid cells. The test item did not show any biologically nor statistically relevant increase in the number of aberrant metaphases. Furthermore, no treatment related increase of endoreduplications or polyploid cells was observed, i.e. neither structural nor numerical aberrations were detected. The positive control test materials, EMS and CPA, induced the expected clear increase in the proportion of cells with chromosomal aberrations. Griseofulvin induced a clear increase in the number of polyploid cells. In conclusion, treatment of V79 cell cultures with the test item, did not increase the proportion of cells with aberrant chromosomes. The test item was not clastogenic in this in vitro test system.

Justification for classification or non-classification

Based on the available data, the test item is not classified and labelled for genototxicity according to Regulation (EC) No 1272/2008.