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Genetic toxicity in vitro

Description of key information

The genotoxic potential of Ethoxy ethoxy ethyl acrylate (CAS No. 7328-17-8) was evaluated in the reverse mutation bavcteria assay (OECD 471) and in the cytogenecity assay evaluating chromosomal aberations 8OECD 473).

In the Ames test, Ethoxy ethoxy ethyl acrylate (CAS No. 7328-17-8) was considered to be non-mutagenic under the conditions of the test.

In the chromosomal aberration test, Ethoxy ethoxy ethyl acrylate (CAS No. 7328-17-8) demonstrated marked toxicity and induced statistically significant increases in the frequency of cells with chromosome aberrations, in both the absence and presence of a liver enzyme metabolizing system (S9) and thus considered to be clastogenic to human lymphocytes in vitro.

Based on these results, the genotoxicity potential needs to be further evaluated in vivo and a testing proposal for chromosal aberrations in vivo (OECD 474) is proposed.

Link to relevant study records

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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:
31 March 2017 - 24 April 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries.
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Principles of method if other than guideline:
NA
GLP compliance:
yes (incl. certificate)
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 M170
- Physical state: Clear colorless liquid
- Analytical purity: 96.20% ( CAS No.: 7328-17-8; EC No.: 230-811-7)
- Impurities (identity and concentrations): Ethyl Carbitol 0.91%, 2-Ethoxy ethyl acrylate(EOEA) 0.21%, EOEOA/AA 1.06%, EOEOEA dimer 1.56%.
- Lot/batch No.: 161225177
- Expiration date of the lot/batch: 24 December 2017
- Storage condition of test material: At ambient temperature (10 to 30 degree celcius) and protected from light (although could be used for formulation in light).
Supplier: Miwon
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 (10% liver S9 in standard co-factors). The S9-mix was prepared before use using sterilized co-factors and maintained on ice for the duration of the test.
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).

Experiment 1: Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Experiment 2: Eight concentrations of the test item (5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Supplier: Fisher Scientific
Batch number (purity): Experiment 1: 1684307 (> 99%) and Expiry 03/22 Experiment 2: 1690734 (> 99%) Expiry 03/22

- Justification for choice of solvent/vehicle: A solubility check showed that 2-phenoxyethyl acrylate was immiscible in sterile distilled water at 50 mg/ml but fully miscible in DMSO at the same concentration.

- Other: 2-phenoxyethyl acrylate was accurately weighed and approximate half-log dilutions prepared in DMSO by mixing on a vortex mixer on the day of each experiment. Formulated concentrations were adjusted to allow for the stated water/impurity content (3.8%)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-Aminoanthracene
Remarks:
Without S9-mix: ENNG, 9AA, 4NQO; With S9-mix: 2AA, BP.
Details on test system and experimental conditions:

Mutation Test - Experiment 1 (plate incorporation method):
Eight concentrations of 2-phenoxyethyl acrylate (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method. 0.1 mL of the appropriate concentration of test item, solvent vehicle or appropriate positive control was added to 2 mL of molten, trace amino-acid supplemented media containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were then mixed and overlayed onto a Vogel-Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.

The same procedure as described above was performed with metabolic activation except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9-mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.

All of the plates were incubated at 37 ± 3oC for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system.
All testing for this experiment was performed in triplicate.

Mutation Test - Experiment 2 (Pre-Incubation Method):
As the result of Experiment 1 was deemed negative, Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation. Seven doses (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, with and without metabolic activation. 0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the test item formulation, solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 oC for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method.

The same procedure as described above was performed with metabolic except that following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9-mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3 oC for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media.

All of the plates were incubated at 37 ± 3 oC for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system.
All testing for this experiment was performed in triplicate.
Evaluation criteria:
Validity criteria:
- negative/positive control data were consistent with historical control data
- positive control showed marked increase over the concurrent negative control
- evaluation was not restricted by loss of plates (e.g. through contamination)
- all bacteria strain must have demonstrated the required charasterics
- the bacterial cell count for each stratin culture should be in the range of 0.9 to 9x109
- there should be a minimum of four non-toxic test item dose levels.
- there should be no evidence of excessive contamination

Evaluation criteria:
- dose-related increases in number of revertant colonies at one or more test points
- increases reproducible between replicate plates
- increases more than twice the corresponding negative control
- biological relevance against in-house historical control ranges
- statistical analysis of data
Statistics:
Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.
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:
The maximum dose level of the test item in the first and second experiment were selected as the maximum recommended dose level of 5000μg/plate. In the first and second mutation test, the test item induced toxicity as a visible reduction in the frequency of revertant colonies of all of the Salmonella tester strains (without a weakening of the bacterial background lawns), at 5000μg/plate in both the presence and absence of metabolic activation (S9-mix).In addition, slight reductions in colony frequency at 1500μg/plate in both the absence and presence of S9-mix. No reductions in colony frequency were noted to Escherichia coli strain WP2uvrA at any test item dose level in either the absence or presence of S9-mix.

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and S9‑mix used in both experiments was shown to be sterile. The culture density for each bacterial strain was also checked and considered acceptable. 

A history profile of vehicle, untreated and positive control values for 2015 and 2016 was included in the test report. The results of this study was in accordance with the history profile.

Conclusions:
The mutagenic potential of Ethoxy ethoxy ethyl acrylate was tested in the reverse mutation in accordance with OECD Test Guideline 471.
Ethoxy ethoxy ethyl acrylate (CAS No. 7328-17-8) was considered to be non-mutagenic under the conditions of this test.
Executive summary:

The mutagenic potential of Ethoxy ethoxy ethyl acrylate was 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 and pre-incubation methods at seven to eight doses (1.5 - 5000 µg/plate), in triplicate, both with and with out present of S9-mix.

The maximum dose level of the test item in the first and second experiment were selected as the maximum recommended dose level of 5000μg/plate. In the first and second mutation test, the test item induced toxicity as a visible reduction in the frequency of revertant colonies of all of the Salmonella tester strains (without a weakening of the bacterial background lawns), at 5000μg/plate in both the presence and absence of metabolic activation (S9-mix). In addition, slight reductions in colony frequency at 1500μg/plate in both the absence and presence of S9-mix. No reductions in colony frequency were noted to Escherichia coli strain WP2uvrA at any test item dose level in either the absence or presence of S9-mix.

Neither experiment one or two showed any increase in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix).

Thus Ethoxy ethoxy ethyl acrylate (CAS No. 7328-17-8) was considered to be non-mutagenic under the conditions of this test.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 March 2017 - 24 April 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
NA
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Principles of method if other than guideline:
NA
GLP compliance:
yes (incl. certificate)
Type of assay:
other: Chromosome aberation test in human lymphocytes (In vitro)
Specific details on test material used for the study:
- Name of test material (as cited in study report): Ethoxy ethoxy ethyl acrylate CAS No. 7328-17-8
- Physical state: Clear colorless liquid
- Analytical purity: 96.20% ( CAS No.: 7328-17-8; EC No.: 230-811-7)
- Impurities (identity and concentrations): Ethyl Carbitol 0.91%, 2-Ethoxy ethyl acrylate(EOEA) 0.21%, EOEOA/AA 1.06%, EOEOEA dimer 1.56%.
- Lot/batch No.: 161225177
- Expiration date of the lot/batch: 24 December 2017
- Storage condition of test material: At ambient temperature (10 to 30 degree celcius) and protected from light (although could be used for formulation in light).
Supplier: Miwon
Target gene:
Structural chromosomal aberrations in cultured mammalian cells, specifically evaluating effects on the metaphase chromosomes of normal human lymphocytes.
Species / strain / cell type:
other: Human Lymphocytes
Details on mammalian cell type (if applicable):
Human lymphocytes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
The dose range for the Preliminary Toxicity Test was 7.35, 14.70, 29.41, 58.81, 117.63, 235.25, 470.5, 941 and 1882 μg/mL. The maximum dose was the maximum recommended dose level, the 10 mM concentration.

No precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure in any of the three exposure groups.

Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 117.63 μg/mL in the 4(20)-hour exposure in the absence of metabolic activation (S9) and 235.25 μg/mL in the 4(20)-hour exposure in the presence of metabolic activation. The maximum dose with metaphases present in the 24-hour continuous exposure was 29.41 μg/mL. The mitotic index data are presented in Table 1 (see attached draft report). The test item induced marked toxicity in all three of the exposure groups.

The selection of the maximum dose level for the Main Experiment was based on toxicity and was 120 μg/mL for the 4(20)-hour exposure in the absence of S9, 360 μg/mL in the presence of S9 and 60 μg/mL for the 24-hour exposure group. The 4(20)-hour exposure group in the presence of S9 was originally performed with a dose range of 60 μg/mL to 480 μg/mL but there were insufficient dose levels suitable for scoring and the exposure was repeated with a revised dose range.

The dose levels used in the main experiments were as follows:
4(20) without S9: 15, 30, 45, 52.5, 60, 75, 120 ug/ml
4(20) with S9 (2%): 7.5, 15, 30, 60, 120, 180, 240, 360 ug/ml
24-hour without S9: 7.5, 15, 30, 37.5, 45, 52.5, 60 ug/ml

Vehicle / solvent:
The test substance was dissolved in dimethyl sulphoxide.
The positive control used in absence of metabolic activation was mitomycin, dissolved in Minimal Essential Medium
The positive control used in the presence of metabolic activation was cyclophosphamide, dissolved in dimethyl sulphoxide.


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:
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non-smoking volunteer (aged 18-35) who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. Based on over 20 years in-house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 hours. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 hours.

Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10 % foetal bovine serum (FBS), at approximately 37 ºC with 5 % CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

The S9 Microsomal fractions were pre-prepared using standardized in-house procedures (outside the confines of this study). Batch Nos. PB/NF S9 31/03/17 and 30/06/17 were used mutagens in the Ames test and the Certificates of S9 Efficacy are presented in Appendix 2 (attached report). The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM). The final concentration of S9, when dosed at a 10% volume of S9-mix into culture media, was 2%.
Rationale for test conditions:
Test conditions validated at the testing facilities in accordance to recognized testing guideline.
Evaluation criteria:
The slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation, if any, of the test item. These observations were used to select the dose levels for mitotic index evaluation. The slides were coded using a computerized random number generator.

A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

Where possible, 300 consecutive well-spread metaphases from each concentration were counted (150 per duplicate), where there were at least 15 cells with aberrations (excluding gaps), slide evaluation was terminated. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing and the ISCN (1985) (Appendix 1). Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides. In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) (including endoreduplicated cells) was also reported.

A test item can be classified as genotoxic if:
1) The number of cells with structural chromosome aberrations is outside the range of the laboratory historical control data.
2) At least one concentration exhibits a statistically significant increase in the number of cells with structural chromosome aberrations compared to the concurrent negative control.
3) The observed increase in the frequency of cells with structural aberrations is considered to be dose-related When all of the above criteria are met, the test item can be considered able to induce chromosomal aberrations in human lymphocytes.



Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test. (Richardson et al. 1989). A toxicologically significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps is less than 0.05 when compared to its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproducible. Incidences where marked statistically significant increases are observed only with gap-type aberrations will be assessed on a case by case basis.
Key result
Species / strain:
other: Human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
Clastogenic to human lymphocytes in vitro
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The molecular weight of the test item was given as 188.22, therefore, the maximum dose level was 1882.2 μg/mL, which was calculated to be equivalent to 10mM, the maximum recommended dose level. The purity of the test item was 96.2% and was accounted for in the test item formulations. The test item was insoluble in culture medium at 18.82 mg/mL but was soluble in dimethyl sulphoxide (DMSO) at 188.2 mg/mL in solubility checks performed in-house.
Prior to each experiment, the test item was accurately weighed, formulated in DMSO and appropriate serial dilutions prepared. There was no significant change in pH when the test item 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 not relevant as formulated within two hours of it being applied to the test system.

- Water solubility: Low water soulbility, therfore dissolved in DMSO.

- Precipitation: No precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure in any of the three exposure groups.

RANGE-FINDING/SCREENING STUDIES: The dose range for the Preliminary Toxicity Test was 7.35, 14.70, 29.41, 58.81, 117.63, 235.25, 470.5, 941 and 1882 μg/mL. The maximum dose was the maximum recommended dose level, the 10 mM concentration. No precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure in any of the three exposure groups. Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 117.63 μg/mL in the 4(20)-hour exposure in the absence of metabolic activation (S9) and 235.25 μg/mL in the 4(20)-hour exposure in the presence of metabolic activation. The maximum dose with metaphases present in the 24-hour continuous exposure was 29.41 μg/mL. The mitotic index data are presented in Table 1. The test item induced marked toxicity in all three of the exposure groups. The selection of the maximum dose level for the Main Experiment was based on toxicity and was 120 μg/mL for the 4(20)-hour exposure in the absence of S9, 360 μg/mL in the presence of S9 and 60 μg/mL for the 24-hour exposure group. The 4(20)-hour exposure group in the
presence of S9 was originally performed with a dose range of 60 μg/mL to 480 μg/mL but there were insufficient dose levels suitable for scoring and the exposure was repeated with a revised dose range.

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 is included in the test report (see attached draft reprt).

Specific details including tables 1 -7 as referenced in the below summary information can be found in attached study report (draft version).

Preliminary toxicity test:

The dose range for the Preliminary Toxicity Test was 7.35, 14.70, 29.41, 58.81, 117.63, 235.25, 470.5, 941 and 1882 μg/mL. The maximum dose was the maximum recommended

dose level, the 10 mM concentration. No precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure in any of the three exposure groups.

Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 117.63 μg/mL in the 4(20)-hour exposure in the absence

of metabolic activation (S9) and 235.25 μg/mL in the 4(20)-hour exposure in the presence of metabolic activation. The maximum dose with metaphases present in the 24-hour continuous

exposure was 29.41 μg/mL. The mitotic index data are presented in Table 1. The test item induced marked toxicity in all three of the exposure groups. The selection of the maximum dose level for the Main Experiment was based on toxicity and was 120 μg/mL for the 4(20)-hour exposure in the absence of S9, 360 μg/mL in the presence of S9 and 60 μg/mL for the 24-hour exposure group. The 4(20)-hour exposure group in the presence of S9 was originally performed with a dose range of 60 μg/mL to 480 μg/mL but there were insufficient dose levels suitable for scoring and the exposure was repeated with a revised dose range.

Main experiment:

The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test. There were metaphases suitable for scoring

present up to 75 μg/mL in the 4(20)-hour exposure in the absence of S9 and up to 30 μg/mL in the 24-hour exposure group. In the presence of metabolic activation (S9), there were metaphases suitable for scoring at the maximum dose level of the test item, 360 μg/mL, however, it was noted that there was an overall reduction in cell numbers at 240 μg/mL and

360 μg/mL. Precipitate observations were made at the end of exposure and no precipitate was observed in any of the three exposure groups.

The mitotic index data for the Main Experiment are given in Table 2 and Table 3. They confirm the qualitative observations in that a dose-related inhibition of mitotic index was

observed. In the 4(20)-hour exposure group in the absence of S9, 61% mitotic inhibition was achieved at 52.5 μg/mL. In the presence of S9, 42% and 37% mitotic inhibition was

achieved at 240 μg/ml and 360 μg/mL, respectively. It was also noted that there were reduced numbers of cells overall at 240 μg/mL and 360 μg/mL, limiting the numbers of metaphases available for scoring although this was not reflected in the mitotic index value. An inhibition of mitotic index of 56% was noted at 30 μg/mL in the 24-hour continuous exposure group.

The maximum dose level selected for metaphase analysis was based on toxicity and was 52.5 μg/mL for the 4(20)-hour exposure in the absence of S9, 360 μg/mL in the presence of

S9 and 30 μg/mL for the 24-hour exposure group. The chromosome aberration data are given in Table 4, Table 5 and Table 6. The assay was considered valid as it met all of the following criteria:

-The frequency of cells with chromosome aberrations (excluding gaps) in the vehicle control cultures were within the current historical control data range.

- All the positive control chemicals induced a demonstrable positive response (p≤0.01) and confirmed the validity and sensitivity of the assay and the integrity of the S9-mix. The ‘A’ replicate of the positive control (CP) in the presence of S9 failed due to a technical error and therefore additional scoring was performed on the partner slide of the ‘B’ replicate. A

significant positive response was demonstrated.

- The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.

- The required number of cells and concentrations were analyzed.

- The test item induced statistically significant increases in the frequency of cells with aberrations in both the absence and presence of metabolic activation.

- The polyploid cell frequency data are given in Table 7. The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in any of

the three exposure groups.

Specifically, the 4(20)-hour exposure group in the absence of S9 achieved marginally greater than acceptable toxicity at the maximum dose level scored and demonstrated a small but

statistically significant response at 52.5 μg/mL. The frequency of the aberrations at 52.5 μg/mL only just exceeded the upper limit of the current historical control range for a vehicle. In the 24-hour exposure group the toxicity achieved at the maximum dose level scored was optimum and a statistically significant response was seen at 30 μg/mL confirming the clastogenicity of the test item in the absence of S9.

The 4(20)-hour exposure group in the presence of S9 demonstrated statistically significant dose related increases in the frequency of cells with aberrations at 180, 240 and 360 μg/mL.

All these dose levels achieved less than optimum toxicity. It was considered that due to the overall reduction in cell numbers seen at 240 and 360 μg/mL that higher dose levels would

not have sufficient metaphases for scoring and the test item was tested to the upper limits of toxicity.

Conclusions:
The clastogenic effetc of Ethoxy ethoxy ethyl acrylate (CAS No. 7328-17-8) was evaluated in human lymphocytes in accordance to OECD 473. All vehicle (dimethyl sulphoxide) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells with aberrations. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Ethoxy ethoxy ethyl acrylate (CAS No. 7328-17-8) demonstrated marked toxicity and induced statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that induced near optimum toxicity.

It was therfore concluded that Ethoxy ethoxy ethyl acrylate CAS No. 7328-17-8 induced statistically significant increases in the frequency of cells with chromosome aberrations, in both the absence and presence of a liver enzyme metabolizing system (S9) c and thus considered to be clastogenic to human lymphocytes in vitro.
Executive summary:

Ethoxy ethoxy ethyl acrylate (CAS No. 7328-17-8) was tested for the potential of inducing chromosome aberrations in cultured mammalian cells in vitro in accordance with the testing guidelines OECD 473 and EC. B10.

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. Three exposure conditions were investigated; 4 hours exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period, 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation.

Based on preliminary toxicity assessment, the dose levels used in the main experiments were as follows:

4(20) without S9: 15, 30, 45, 52.5, 60, 75, 120 ug/ml

4(20) with S9 (2%): 7.5, 15, 30, 60, 120, 180, 240, 360 ug/ml

24-hour without S9: 7.5, 15, 30, 37.5, 45, 52.5, 60 ug/ml

A dose-related inhibition of the mitotic index was observed. In the 4(20)-hour exposure group in the absence of S9, 61% mitotic inhibition was achieved at 52.5 μg/mL. In the presence of S9, 42% and 37% mitotic inhibition was achieved at 240 μg/ml and 360 μg/mL, respectively. An inhibition of mitotic index of 56% was noted at 30 μg/mL in the 24-hour continuous exposure group.

The maximum dose level selected for metaphase analysis was based on toxicity and was 52.5 μg/mL for the 4(20)-hour exposure in the absence of S9, 360 μg/mL in the presence of

S9 and 30 μg/mL for the 24-hour exposure group. Specifically, the 4(20)-hour exposure group in the absence of S9 achieved marginally greater than acceptable toxicity at the maximum dose level scored and demonstrated a small but statistically significant response at 52.5 μg/mL. The frequency of the aberrations at 52.5 μg/mL only just exceeded the upper limit of the current historical control range for a vehicle. In the 24-hour exposure group the toxicity achieved at the maximum dose level scored was optimum and a statistically significant response was seen at 30 μg/mL confirming the clastogenicity of the test item in the absence of S9. The 4(20)-hour exposure group in the presence of S9 demonstrated statistically significant dose related increases in the frequency of cells with aberrations at 180, 240 and 360 μg/mL.

All vehicle (dimethyl sulphoxide) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells with aberrations. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

In conclusion, Ethoxy ethoxy ethyl acrylate (CAS No. 7328-17-8) demonstrated marked toxicity and induced statistically significant increases in the frequency of cells with aberrations in both the absence and presence of a liver enzyme metabolizing system (S9), using a dose range that included a dose level that induced near optimum toxicity. Thus, Ethoxy ethoxy ethyl acrylate CAS No. 7328-17-8 are considered to be clastogenic to human lymphocytes in vitro.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

No in vivo studies performed. Based on available results from in vitro studies, a testing proposal for chromosal aberrations in vivo (OECD 474) is proposed.

Endpoint conclusion
Endpoint conclusion:
no study available (further information necessary)

Additional information

Justification for classification or non-classification

Based on available in vitro data, no classification is proposed. The positive findings in vitro where Ethoxy ethoxy ethyl acrylate (CAS No. 7328-17-8) demonstrated marked toxicity and induced statistically significant increases in the frequency of cells with chromosome aberrations, in both the absence and presence of a liver enzyme metabolizing system (S9) and thus was considered to be clastogenic to human lymphocytes in vitro, will be further evaluated in vivo. Thus an in vivo chromosome aberration tests (OECD 474) has been propose as testing proposal.