Biphenyl-2-ol, ethoxylated, esters with acrylic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

 

Mutagenicity, in vitro, Salmonella typhimurium and Escherichia Coli:             

OECD 471 testing performed with Biphenyl-2-ol, ethoxylated, esters with acrylic acid was negative for mutagenic effects both with and without metasbolic activation..

 

Mutagenicity, in vitro, mutations mammalian cells:

No data is available on Biphenyl-2-ol, ethoxylated, esters with acrylic acid.

 

OECD 476 testing performed with 2-phenoxyethyl acrylate was negative for mutagenic effects both in with and without metabolic activation. Due to the very close structural similarity to2-phenoxyethyl acrylate, the target substance o-phenylphenolethyl acrylate should be considered without mutagenic potential in mammalian cells in vitro as well.  

 

Mutagenicity, in vitro, chromosome aberration mammalian cells:

 

OECD 473 tests performed with 2-phenoxyethyl acrylate as well as with the target substance Biphenyl-2 -ol, ethoxylated, esters with acrylic acid were negative both with and without metabolic activation. These comparable data further supports the read-across approach for the toxicological end-points.

 

Overall, these in vitro data indicate that biphenyl-2 -ol, ethoxylated, esters with acrylic acid can be considered as having no mutagenic or clastogenic potential.

 See justification for read-across attached in section 13.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

The hypothesis for a read-across approach from data on 2-phenoxyethyl acrylate to o-phenylphenolethyl acrylate is the similarity of the chemical structures and physical chemical properties of the two substances.
See attached read-across justification in section 13

Reason / purpose for cross-reference:

read-across source

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Strain/cell type

Remarks:

Migrated from field 'test system'

Executive summary:

No data is available for Biphenyl-2-ol, ethoxylated, esters with acrylic acid (M1142) (T).

 

2-phenoxyethyl acrylate was tested in vitro in a mammalian cell gene mutation test using mouse lymphoma L5178Y cells in accordance with OECD test guideline 476. 2-phenoxyethyl acrylate did not induce dose-related increases in the mutant frequency at any condition. Hence, 2-phenoxyethyl acrylate was considered to be non-mutagenic under the conditions of the test. Due to structural and pysico-chemical similarity to Biphenyl-2-ol, ethoxylated, esters with acrylic acid, a non-mutagenic potential can also be concluded for Biphenyl-2-ol, ethoxylated, esters with acrylic acid as well.

 

See justification for read-across attached in section 13.

Reference 2

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

Study period:

2008

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: TCCA(Toxic Chemical Control Act)- Test Guidelines described in Annex 5 of TCCAGood Laboratory Practice Standards and Test Guidelines” Notification No. 2006-29, National Institute of Environmental Research, Korea (Dec. 19, 2006)

Deviations:

no

Principles of method if other than guideline:

NA

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: Chinese Hamster Lung (CHL) cell line

Target gene:

Structural chromosomal aberrations in cultured mammalian cells

Species / strain / cell type:

mammalian cell line, other: Chinese Hamster Lung (CHL) cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Chinese Hamster Lung (CHL) cell line passage 20, 29 and 34.
- Suitability of cells: Guideline approved cell line with large historical database.
- Cell cycle length, doubling time or proliferation index: Chromosome number of chromosome was 25 per cell, and the cell doubling time was about 15 hours.
- Sex, age and number of blood donors if applicable: NA
- Whether whole blood or separated lymphocytes were used if applicable: NA
- Number of passages if applicable: NA
- Methods for maintenance in cell culture if applicable: DMEM 10% FBS (heat inactivated) 1% pen-strep, in incubator 37 degrees Celcius, 5% CO2.
- Modal number of chromosomes: 25/cell
- Normal (negative control) cell cycle time:

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mixture

Test concentrations with justification for top dose:

A cytotoxicity test was performed to determine the concentration of the test item to be used in the chromosome aberration test. Following concentration range was used (2 – 2628.7µg/mL). In the presence of S9-mix IC50 was estimated to 125.64 µg/mL and in absence of S9-mix and short treatment period IC50 was estimated to 17 µg/mL and during continuous treatment estimated to 7.91 µg/mL.

The highest concentration used for the chromosome aberration test were determined 130µg /mL

The dose levels used in the main experiments were selected using data from the cytotoxicity test and were as follows:

Short treatment (6hours incubation and 18 hours expression)
- Concentration: 5, 10 and 20µg/mL, with S9-mix.
- Concentration: 32.5, 65 and 130µg/mL, without S9-mix.

Continuous treatment (24 hours treatment and expression)
- Concentration: 2.5, 5 and 10µg/mL, with S9-mix.

Confirmation study (6 hours treatment)
- Concentration: 110, 120 and 130µg/mL, without S9-mix.

All plates included a negative control (1% DMSO) and positive control (0.2µg/mL MMC or 5µg/mL CP).

Vehicle / solvent:

The test substance was dissolved in dimethyl sulphoxide.
The positive control used in absence of metabolic activation was mitomycin C (MMC), dissolved in Minimal Essential Medium
The positive control used in the presence of metabolic activation was cyclophosphamide monohydrate (CP), dissolved in Minimal Essential Medium

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Suspended CHL cells were seeded 2x104 cell/5mL in a 60 mm petri dish and cultured as monolayer at 37C with 5% CO2 in humified air. Cell medium used was Eagles minimal essential medium with 1% penicilin/streptomycin, and 10% foetal bovine serum (FBS)

SPINDLE INHIBITOR (cytogenetic assays): Incubated with colcemid (0.25 mg/mL) for 2 hours

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: not specified

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Cells were incubated in 0.075 M KCL solution at 37C, for 30 minutes. Then cells were fixed in a methanol – acid solution (3:1), air-dried and stained using 5% Giemsa solution for 10 minutes, and the washed.

NUMBER OF CELLS EVALUATED: 2 x 104 cells in 5mL

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 200 metaphase cells/sample

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: MTT assy used for measure cell viability. Cell viability = (test item treated cells/ negative control cells) x 100 (%)

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- OTHER: S9 metabolic enzymes were imported and used to Sprague-Dawley rats induced with the
Aroclor 1254 separated from MOLTOX postmitochondrial supernatant (Lot. 2191, Molecular
Toxicology, Inc.). S9 mixture was prepared as 10% of the culture volume.

Evaluation criteria:

Chromosome aberration test was determined satisfying if the results were following determination of reference.


1) The negative control group treated with solvent, the number of chromosome abnormalities in
cells should be within the following ranges of historical data.

- Without S9 mix : 200 of the metaphase cell min=0, max=6(mean=1.5, SD=0.8), not
including the gap.
- With S9 mix : 200 of the metaphase cell min=0, max=3(mean=0.8, SD=0.4), not including
the gap.

2) Positive control a significantly increased number of chromosome aberrations
(Fisher exact test, P<0.05).

3) Chromosomal aberration was classified in the „Edition Atlas Chromosomal aberration(1988)‟

4) Test results and evaluation:
- Chromosomal aberration marked in 200 metaphase cells for each concentration.
- A structural aberration was conducted that it was classified as chromosome structure break
and exchange, chromatid break and exchange. Gap was classified as defects than the with
of a narrow gap chromatid and counted, but not included in the chromosomal aberration.
More than four in number than diploid and polyploidy endoreduplication only evaluated.
- Numerical aberration was evaluated only polyploidy and endoreduplication as 4 diploid
aberration.
- A cell having a structure in one or more types of one or more positive cells, and obtains
the percentage by count, it was determined this to chromosome aberration frequency. In
addition, the type of chromosomal aberration was recorded, respectively.

Statistics:

Statistical analysis:

- Statistical analysis for chromosome aberration frequency was conducted in a number that
does not include a gap depending on the OECD guide line.
- The number of Chromosomal aberration and normal in 200 metaphase each group cell was
marked.
- To indicate the does-dependent increase or repeated increase was judge as positive.
- Through the Fisher‟s exact test (Altman, 1993) was performed for statistical significance
for the negative control group and the test substance-treated group. According to P-value,
P<0.05 was determined whether when positive.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

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: 2-phenoxyethyl acrylate was accurately weighed, dissolved in dimethyl sulphoxide and serial dilutions prepared. The molecular weight of the test item was given as 192.2, therefore the maximum dose level was 1922 μg/ml, which was calculated to be equivalent to the 10 mM maximum recommended dose level. The purity of the test item was 85.5% and was accounted for in the formulations. There was no significant change in pH when dissolved into dimethyl
sulphoxide.


- Evaporation from medium: No information but coinsidered not relevant. o-Phenylphenol ethyl acrylate was formulated within a few hours of it being applied to the test system.

- Water solubility: non-soulble in water, therfore dissolved in DMSO.

- Precipitation:No precipitate observed

RANGE-FINDING/SCREENING STUDIES: A preliminary toxicity test was performed to determine the concentration of the test item to be used in the chromosome aberration test. Following concentration range was used (2 – 2628.7µg/mL). In the presence of S9-mix IC50 was estimated to 125.64 µg/mL and in absence of S9-mix and short treatment period IC50 was estimated to 17 µg/mL and during continuous treatment estimated to 7.91 µg/mL.

COMPARISON WITH HISTORICAL CONTROL DATA: All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated. Historical contro data for the vehicle and the positive control was included in the test report.

Conclusions:

The mutagenic potential of o-Phenylphenol ethyl acrylate was tested In Vitro Chromosome Aberration Test of using Mammalian Culture Cell in accordance with OECD Test Guideline 473. The test item, o-Phenylphenol ethyl acrylate, did not induce the numerical and structural chromosome aberrations in Chinese Hamster Lung cells under the conditions of this study.

Executive summary:

The mutagenic potential of o-Phenylphenol ethyl acrylate was tested in an In Vitro Chromosome Aberration Test of using Mammalian Cells in accordance with OECD Test Guideline 473.

From a preliminary cytotoxicity test (using conc. 2- 2628.7 µg/mL) 130µg/mL was selected as the highest concentration for testing the ability of o-Phenylphenol ethyl acrylate to induce chromosome aberrations in Chinese Hamster Lung (CHL) cells. CHL cells were treated for 6 hours with 32.5, 65, or 130 µg/mLo-Phenylphenol ethyl acrylate with or without metabolic activation S9-mix. The number of chromosome aberration in cells excluding gaps significantly increased after 6 hours treatment with the highest dose (130 µg/mL) including S9-mix. However, the increase was due to 33 % viability in chromosomal aberration and could not be reproduced in the following confirmation test with an identical setup.

24 hours of treatment with 2.5, 5, or 10 µg/mLo-Phenylphenol ethyl acrylate with or without metabolic activation S9-mix did not induce chromosome aberration.

The negative and positive controls were within the range of historical data confirming the validity of the tests.

 

In conclusion:

The test item,o-Phenylphenol ethyl acrylate, did not induce the numerical and structural chromosome aberrations in CHL cells under the conditions of this study.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: genome mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012.07.02 - 2012.09.17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study performed in accordance with recognized testing guidelines with no deviations.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

NA

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase gene (TK+/-)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/ml), Streptomycin (100 μg/ml), Sodium pyruvate (1 mM), Amphotericin B (2.5 μg/ml) and 10% donor horse serum (giving R10 media).RPMI 1640 with 20% donor horse serum (R20) and without serum (R0) were used during the course of the study

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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 was prepared from the livers of male Sprague-Dawley rats. These had each received, orally, three consecutive daily doses of phenobarbital/b-naphthoflavone (80/100 mg per kg per day) prior to S9 preparation on the fourth day.

Test concentrations with justification for top dose:

Preliminary toxicity test: 0, 7.51, 15.02, 30.03, 60.06, 120.13, 240.25, 480.5, 961, 1922 μg/ml.
Mutation tests-experiment I: 0, 1.25,2.5, 5, 10, 20, 25, 30 μg/ml.
Mutation tests-experiment II: 0, 0.63, 1.25, 2.5, 5, 10, 20, 30, 40 μg/ml.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: Following solubility checks, DMSO was chosen as vehicle

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Details on test system and experimental conditions:

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with 2-phenoxyethyl acrylate at eight dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, due to the equivocal response observed
in the presence of metabolic activation in Experiment 1, the cells were treated with 2-phenoxyethyl acrylate at eight dose levels using a repeat of the 4-hour exposure group in the presence of metabolic activation (2% S9) and a 24-hour exposure group in the absence of metabolic activation.

At the end of the treatment period, for each experiment, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 x 105 cells/ml. The cultures were incubated at 37°C with 5% CO 2 in air and subcultured every 24 hours for the expression period of two days by counting and diluting to 2 x 105 cells/ml.

On Day 2 of the experiment, the cells were counted, diluted to 104 cells/ml and plated for mutant frequency (2000 cells/well) in selective medium containing 4 μg/ml 5-trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/ml and plated (2 cells/well) for viability (%V) in non-selective medium.

The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post treatment toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data a Relative Total Growth (RTG) value.

Microtitre plates were scored using a magnifying mirror box after ten to fourteen days’ incubation at 37°C with 5% CO 2 in air. The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutation plates were also recorded. Colonies are scored manually by eye using qualitative judgement. Large colonies are defined as those that cover approximately ¼ to ¾ of the surface of the well and are generally no more than one or two cells thick. In general, all colonies less than 25% of the average area of the large colonies are scored as small colonies. Small colonies are normally observed to be more than two cells thick. To assist the scoring of the TFT mutant colonies 0.025 ml of thiazolyl blue tetrazolium bromide (MTT) solution, 2.5 mg/ml in phosphate buffered saline (PBS), was added to each well of the mutation
plates. The plates were incubated for approximately two to three hours. MTT is a vital stain that is taken up by viable cells and metabolised to give a brown/black colour, thus aiding the visualisation of the mutant colonies, particularly the small colonies.

Based on the scoring, calculation of percentage Relative Suspension Growth (%RSG), Day 2 Viability (%V), Relative Total Growth (RTG) and the Mutation Frequency (MF) was performed.

Evaluation criteria:

A mutagenic is concluded when a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value is observed. Following discussions at an International Workshop on Genotoxicity Test Procedures in Plymouth, UK, 2002 (Moore et al 2003) it was felt that the IMF must exceed some value based on the global background MF for each method (agar or microwell). This Global Evaluation Factor (GEF) value was set following a further meeting of the International Workshop in Aberdeen, Scotland, 2003 (Moore et al 2006) at 126 x 10-6 for the microwell method.

Therefore, any test item dose level that has a mutation frequency value that is greater than the corresponding vehicle control by the GEF of 126 x 10-6 and demonstrates a positive linear trend will be considered positive. However, if a test item produces a modest increase in mutant frequency, which only marginally exceeds the GEF value and is not reproducible or part of a dose-related response, then it may be considered to have no toxicological significance. Conversely,
when a test item induces modest reproducible increases in the mutation frequencies that do not exceed the GEF value then scientific judgement will be applied. If the reproducible responses are significantly dose-related and include increases in the absolute numbers of mutant colonies then they may be considered to be toxicologically significant.

Small significant increases designated by the UKEMS statistical package was reviewed using the above criteria, by the Study Director and could be disregarded at the Study Director's discretion.

Statistics:

UKEMS statistical package

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:There was no marked change in pH when 2-phenoxyethyl acrylate was dosed into media.
- Effects of osmolality: The osmolality did not increase by more than 50 mOsm (Scott et al 1991).
- Evaporation from medium: No information but considered to not relevant.
- Water solubility: Formulations prepared in DMSO, not soluble in water.
- Precipitation: The maximum proposed dose level in the solubility test was set at 1922 μg/ml, the approximate 10 mM limit dose. The purity of 2-phenoxyethyl acrylate was 85.5% and was therefore accounted for when formulating the dosing solutions.

RANGE-FINDING/SCREENING STUDIES: A preliminary toxicity test was performed on cell cultures at 5 x 105 cells/ml, using a 4-hour exposure period both with and without metabolic activation (20% S9-mix), and at 1.5 x 105 cells/ml using a 24-hour exposure period without metabolic activation. The dose range used in the preliminary toxicity test was 7.51 to 1922 μg/ml for all three of the exposure groups.

COMPARISON WITH HISTORICAL CONTROL DATA: The vehicle and positive control data were in accordance with the historical Vehicle and Positive Control Mutation Frequencies obtained by the testing laboratorie (Harlan Laboratories).

Remarks on result:

other: strain/cell type:

Remarks:

Migrated from field 'Test system'.

The maximum dose levels used in the Mutagenicity Test were limited by test item-induced toxicity. Precipitate of test item was not observed at any of the dose levels in the Mutagenicity Test. The vehicle (solvent) controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system. 2-phenoxyethyl acrylate did not induce any reproducible toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment.

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

2-phenoxyethyl acrylate was non-mutagenic and did not induce any clastogenic effects to L5178Y mouse lymphoma cells treated in vitro.

Executive summary:

2-phenoxyethyl acrylate was tested in vitro in a mammalian cell gene mutation test using mouse lymphoma L5178Y cells (OECD 476). Two independent experiments were performed. In Experiment 1, mouse lymphoma cells were treated with 2-phenoxyethyl acrylate at eight dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, due to the equivocal response observed in the presence of metabolic activation in Experiment 1, the cells were treated with 2-phenoxyethyl acrylate at eight dose levels using a repeat of the 4-hour exposure group in the presence of metabolic activation (2% S9) and a 24-hour exposure group in the absence of metabolic activation.

The dose range was selected following the results of a preliminary toxicity test and was 1.25 to 30 μg/ml in the absence of metabolic activation, and 7.5 to 240 μg/ml in the presence of metabolic activation for Experiment 1. In Experiment 2 the dose range was 0.63 to 40 μg/ml in the absence of metabolic activation, and 60 to 240 μg/ml in the presence of metabolic activation.

The maximum dose levels used in the Mutagenicity Test were limited by test item-induced toxicity. Precipitate was not observed at any of the dose levels in the Mutagenicity Test. The vehicle (solvent) controls had mutant frequency values that were considered acceptable. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

2-phenoxyethyl acrylate did not induce any reproducible toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment. Hence, 2-phenoxyethyl acrylate was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 November 2007 - 11 February 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: TCCA(Toxic Chemical Control Act)- Test Guidelines described in Annex 5 of TCCAGood Laboratory Practice Standards and Test Guidelines” Notification No. 2006-29, National Institute of Environmental Research, Korea (Dec. 19, 2006)

Principles of method if other than guideline:

NA

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Name of test material (as cited in study report): MIRAMER OPPEA
- Physical state: liquid, acrylic mild odor
- Analytical purity: APPEA 90.50% (CAS No.: 72009-86-0)
- Impurities (identity and concentrations): OPPEO (2.2%), OPP(EO)2 (1.3%), OPP(EO)2A (5.5%).
- Lot/batch No.: 71016M95
- Expiration date of the lot/batch: Not specified
- Storage condition of test material: Room temperature (normal temperature).
- Supplier: Miwon Commercial Co., Ltd.

Target gene:

Strain Target mutation Mutation type
TA 1535 hisG46; rfa-; uvrB- Base-pair substitution
TA 100 hisG46; rfa-; uvrB-; R-factor Base-pair substitution
TA 98 hisD3052; rfa-; uvrB-, R-factor Frame shift
TA 1537 hisC3076; rfa-; uvrB-; Frame shift
WP2uvrA trp-, urvA- Base-pair substition

All of the Salmonella strains are histidine dependent by virtue of a mutation through the histidine operon and are derived from S. typhimurium strain LT2 through mutations in the histidine locus. Additionally, due to the "deep rough" (rfa-) mutation they possess a faulty lipopolysaccharide coat to the bacterial cell surface thus increasing the cell permeability to larger molecules. A further mutation, through the deletion of the uvrB-bio gene, causes an inactivation of the excision repair system and a dependence on exogenous biotin. In the strains TA98 and TA100, the R-factor plasmid pKM101 enhances chemical and UV-induced mutagenesis via an increase in the error prone repair pathway. The plasmid also confers ampicillin resistance which acts as a convenient marker (Mortlemans and Zeiger (2000)). In addition to a mutation in the tryptophan operon, the E. coli tester strain contains a uvrA- DNA repair deficiency which enhances its sensitivity to some mutagenic compounds. This deficiency allows the strain to show enhanced mutability as the uvrA repair system would normally act to remove and repair the damaged section of the DNA molecule (Green and Muriel (1976)).

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

NA

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

rat liver homogenate metabolizing system (5% liver S9 in standard co-factors).

Test concentrations with justification for top dose:

The test item was tested using the following method with and without metabolic activation. The maximum concentration was 5000 µg/plate (the maximum recommended dose level), a positive and a negative control were included on each plate.

Dose range finding study: Seven concentrations of the test item including highest dose level by
applying a common ratio of 4 (1.22, 4.88, 19.5, 78.1, 312.5, 1250 and 5000 µg/plate) ) were assayed in triplicate against each tester strain, using the streak plate method.

Main study: Six concentrations of the test item (156.3, 312.5, 625, 1250, 2500, 5000 µg/plate) were assayed in triplicate against each tester strain, using the streak plate method.

Confirmation study: Six concentrations of the test item (156.3, 312.5, 625, 1250, 2500, 5000 µg/plate) were assayed in triplicate against each tester strain, using the streak plate method.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (Cas. No. 67-68-5)
Supplier: Sigma-Aldrich, U.S.A.
Batch number (purity): 105K00431
- Justification for choice of solvent/vehicle: o-phenylphenol ethyl acrylate was immiscible in water but fully miscible in DMSO at the same concentration.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-Aminoanthracene and 2-Amonofluorene

Remarks:

Without S9-mix: 2-AA; With S9-mix: 2-NF; SA; 9-AA; AF-2.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation)
- Cell density at seeding (if applicable): NA

- Test method:
1) Master plate made from cryogenic freezing strains.
2) A single colony from the master plate was inoculated into 10ml of the 1.6% NB medium and was incubated for 12 ~ 14 hours at 37 0C, 200 rpm.
3) 0.1 mL of the test substance, 0.1 mL of fresh culture bacteria, 0.5 mL of S9 mix or 0.2 M SPB were placed in respective sterile tubes. Top agar were added to 2 mL respectively and then they were mixed with a vortex mixer. This mixture was poured on the minimum
glucose agar plates and allowed to solidify.
4) After the top agar was solidified, the plates were inverted and cultured in an incubator at 37°C for approximately 48 hours, and count the colony.
5) In order to confirm the microbial contamination, 0.1 mL of the highest dose test substance, 0.5 mL of S9 mixture and 0.2 M SPB were placed in sterilized test tubes, added to 2 mL of top agar and mixed with a vortex mixer after culture.
6) Three plates per dose were used.
7) Plates was lowered the biased tendencies selecting a random.
8) It writes information of test number, name of strain, concentration, S9 the mixture presence or absence on a plate with a lid

- Medium:
1) Vogel-Bonner minimal nutrient agar plate:
Suitable Vogel-Bonner E medium was added to 1.5% bacto agar and autoclaved. After sterilized, 2% D-glucose were added. The mixed solution was transferred to petri dishes (30L) and allowed to solidify room temperature.
2) Top agar plate:
Bacto agar and sodium chloride were weighed. Distilled water was added to yield the concentration of 0.6 and 0.5% w/v, respectively and then autoclaved. And before use just prior to use the addition of 0.5 mM L-Histidine / D-Biotin or and 0.5 mM L-Tryptophan / 0.5 mM L-Histidine / D-Biotin solution to10ml in 45 0C

Evaluation criteria:

Validity criteria:
- negative/positive control data were consistent with historical control data.

Evaluation criteria:
1) S9 (-) or S9 (+) condition is repeatable increase in dose dependent increase or a capacity
more than the capacity of reverse mutation colonies on at least one strain or observed in the
case that the significant linear relationship.

However, the statistical result is not the determining factor, should be considered the
biological validly of the results. Therefore, it may be used in the following evaluation
standard.

2) The number of reverse mutation colonies is than two times the control group of TA98,
TA100 and WP2 uvrA strains. TA1535 and TA1537, the strain is more than three times, the
dose-response relationship, and may be positive, when the recognition reproducibility

Statistics:

Summary statistics: mean and standard deviation (SD). No statistical test performed.

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

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:

valid

Positive controls validity:

valid

Additional information on results:

Dose range finding study:
High dose of the test article was selected as 5000 µg/plate as recommended in the guideline. This high dose was sequentially diluted by 7 dose levels (1.2 ~ 5000 µg/plate). Growth inhibition of the test item was evident at TA98 and TA100 in absence of metabolic activation. However, growth inhibition of the test article was not evident in presence of metabolic activation. Also, precipitate was observed at 1250, 2500 µg/plate concentration of the test item.

Main study:
When the TA98, TA100, TA1535, TA1537 and WP2 uvrA strain were exposed to the test substance (156.3 ~ 5000 µg/plate) in absence of S-9 mix, dose dependent increase in the number of mutant colonies was not observed. In the test system that was added to the S9 mix, dose dependent increase in all concentrations of the test substance was not observed. Precipitation was observed in 5000 µg/plate concentration, but
it did not affect colony count. Precipitation was observed in 5000 µg/plate concentration without affecting colony count. The numbers of revertant colonies in negative control groups were within the range of the historical control data. The numbers of revertant colonies in the positive control groups were remarkably increased the compared to negative control group with S9 mix and without S9 mix. Therefore, It proved the validity and activity of the S9 mix of the test.

Confirmation study:
To confirm the experimental results, using the same concentration as the test was performed. When exposed to the test substance (156.3 ~ 5000 µg/plate) in the TA98, TA100, TA1535, TA1537 and WP2 uvrA strain without S9 mix condition, dose dependent increase in the number of mutant colonies was not observed. In the test system was added to the S9 mix, dose dependent increase in all concentrations of the test substance was not observed.

A history profile of vehicle, untreated and positive control values for 2006 was included in the test report. The results of this study was in accordance with the history profile.

Conclusions:

The mutagenic potential ofo-Phenylphenol ethyl acrylate was tested in the reverse mutation in accordance with OECD Test Guideline 471.
o-Phenylphenol ethyl acrylate was found to be non-mutagenic in the reverse mutation assay (Ames Test) using Salmonella Typhimurium and Escherichia Coli with and without metabolic activation.

Executive summary:

The mutagenic potential of o-Phenylphenol ethyl acrylatewas tested in the reverse mutation assay (Ames Test) using Salmonella Typhimurium strains (TA1535, TA1537, TA98, TA100) and Escherichia Coli strain (WP2uvrA). The test in accordance with OECD Test Guideline 471 using the plate incorporation methods at six to eight doses (156.3 - 5000 µg/plate), in triplicate, both with and without present of metabolic activation (S9-mix).

The maximum dose level of the test item in the three experiments (preliminary-, main-, and confirmatory experiment) were selected as the maximum recommended dose level of 5000μg/plate

No significant increase in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of o-Phenylphenol ethyl acrylate, with or without S9 mix.

Thus o-Phenylphenol ethyl acrylate was considered to be non-mutagenic under the conditions of this test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

The available in vitro data indicate that Biphenyl-2-ol, ethoxylated, esters with acrylic acid should not be classified as mutagenic or genotoxic.