2-Propenoic acid, 2-[2-(ethenyloxy)ethoxy]ethyl ester

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The following studies are  available, all of which gave negative results.

- ames study ( bacterial reverse mutation assay)

- in-vitro chromosome aberration

- in-vitro mouse lymphoma assay

A positive result was produced in a second in-vitro Chromosome Aberration studies, this possibly occurred due to a one (or more ) of the following reasons:

-       The chinese hamster lung V79 cell line was used, this is recognised for providing positive results, and due to this, is not used very often.

-        The second test was not performed due to clastogenicity after 24 hours

-       The dose used for without S9 mix was too high and could have contributed to this result.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

22/02/06 - 03/04/06

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of relevant ressults.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 22/07/2004 Date of signature: 06/01/2005

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/beta-naphthaflavone

Test concentrations with justification for top dose:

-S9: 3.2, 6.4, 12.9, 25.8, 51.6 and 103.1 µg/ml

+S9: 51.6, 103.1, 206.3, 412.5, 825.0 and 1650.0 µg/ml

Untreated negative controls:

yes

Remarks:

0 µg/ml

Negative solvent / vehicle controls:

yes

Remarks:

solvent treatment groups were used as the vehicle control

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation

Untreated negative controls:

yes

Remarks:

0 µg/ml

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

The test item 2-(2-Vinyloxyethoxy)ethyl acrylate, dissolved in deionised water, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in one experiment. The following study design was performed: With and without S9 mix Exposure period 4 hrs Recovery 14 hrs Preparation interval 18 hrs In each experimental group two parallel cultures were set up. Per culture at least 100 metaphase plates were scored for structural chromosome aberrations. The highest applied concentration in the pre-test on toxicity (1652 Ng/mL; approx. 8.9 mM) was chosen with regard to the solubility properties of the test item in an appropriate solvent with respect to the current OECD Guideline 473. Dose selection for the cytogenetic experiments was performed considering the toxicity data. The chosen treatment concentrations are described in Table 2 (page 16). The evaluated experimental points and the results are summarised in Table 1 (page 10). In the absence of S9 mix, cytotoxicity was observed at the highest evaluated concentration. In the presence of S9 mix, concentrations showing clear cytotoxicity were not scorable for cytogenetic damage. In the absence and presence of S9 mix, statistically significant and biologically relevant increases in the number of cells carrying structural chromosomal aberrations were observed after treatment with the test item. In addition, in the absence of metabolic activation the number of aberrant cells were increased in a dose-related manner. No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls. Appropriate mutagens were used as positive controls. They induced statistically significant increases (p<0.05) in cells with structural chromsome aberrations.

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported, the test item induced structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro. Therefore, 2-(2-Vinyloxyethoxy)ethyl acrylate is considered to be clastogenic in this chromosome aberration test in the absence and presence of S9 mix.

Executive summary:

This study followed the procedures indicated by the following internationally accepted guidelines and recommendations: Ninth Addendum to the OECD Guidelines for Testing of Chemicals, February 1998, adopted July 21, 1997, Guideline No. 473 "In vitro Mammalian Chromosome Aberration Test". Commission Directive 2000/32/EC, L1362000, Annex 4A: "Mutagenicity – In vitro Mammalian Chromosome Aberration Test", dated May 19, 2000. Japanese Guidelines: "Kanpoan No. 287 -- Environmental Agency" "Eisei No. 127 -- Ministry of Health & Welfare" ”"eisei 09/10/31 Kikyoku No. 2 -- Ministry of International Trade & Industry".

The test item 2-(2-Vinyloxyethoxy)ethyl acrylate, dissolved in deionised water, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in one experiment. The following study design was performed:

With and without S9 mix: Exposure period 4 hrs; Recovery 14 hrs; Preparation interval 18 hrs

In each experimental group two parallel cultures were set up. Per culture at least 100 metaphase plates were scored for structural chromosome aberrations. The highest applied concentration in the pre-test on toxicity (1652 Ng/mL; approx. 8.9 mM) was chosen with regard to the solubility properties of the test item in an appropriate solvent with respect to the current OECD Guideline 473. Dose selection for the cytogenetic experiments was performed considering the toxicity data. In the absence of S9 mix, cytotoxicity was observed at the highest evaluated concentration. In the presence of S9 mix, concentrations showing clear cytotoxicity were not scorable for cytogenetic damage. In the absence and presence of S9 mix, statistically significant and biologically relevant increases in the number of cells carrying structural chromosomal aberrations were observed after treatment with the test item. In addition, in the absence of metabolic activation the number of aberrant cells were increased in a dose-related manner. No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls. Appropriate mutagens were used as positive controls. They induced statistically significant increases (p<0.05) in cells with structural chromsome aberrations.

In conclusion, it can be stated that under the experimental conditions reported, the test item induced structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro. Therefore, 2-(2-Vinyloxyethoxy)ethyl acrylate is considered to be clastogenic in this chromosome aberration test in the absence and presence of S9 mix.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between Feb. 23rd and May 25, 2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete or methodological deficiencies, which do not affect the quality of relevant results

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

see priniciples of method

Principles of method if other than guideline:

The wells were not washed after 4 hours in culture in the growth inhibition test. In this study, it was found that the test substance was dissolved into the vehicle and not precipitated. Moreover, the results of the absorbance of negative control were within the range of the standard deviation of the historical control dtaa in our laboratory. Therefore protocol deviation noted above was not considered to give an effect on the integrity and interpretation of the data or outcome of the study.

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: not stated Date of Signature: 25th May 2006

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable.

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Model chromosome number of Chinese hamster lung (CHL/IU) cells is 25, and cell-cycle is 15-17 hours. Cells were cultured with EMEM in the C02 incubator.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (rat induced liver PB + BF)

Test concentrations with justification for top dose:

A growth inhibition test was conducted in 9 dose levels, in which the highest dose of 5000 µg/mL was sequentially diluted to 8 additional lower doses (2.5, 5, 10, 105, 210, 420µg/mL) with the negative control groups.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO, Lot No. : K33960231 504, Merck, USA)
- Justification for choice of solvent/vehicle: Dimethyl sulfoxide (DMSO) was used as a vehicle since the test substance was dissolved through the pre-preparation of the test substance.

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

In the presence of S9 Migrated to IUCLID6: (CP)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

In the absence of S9 Migrated to IUCLID6: (MMC)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
in medium


DURATION
- Preincubation period:
48 hrs

- Exposure duration:
Short term exposure = 6 hours Continuous treatment = 24 hours

- Expression time (cells in growth medium):
18 hrs for 6 hrs exposure.

- Selection time (if incubation with a selection agent):
Not applicable.

- Fixation time (start of exposure up to fixation or harvest of cells):
24 hrs.


SELECTION AGENT (mutation assays):
No selection agent.

SPINDLE INHIBITOR (cytogenetic assays): Colcemid


STAIN (for cytogenetic assays):
Stained with 5% Giemsa solution


NUMBER OF REPLICATIONS:
Duplicate cultures


NUMBER OF CELLS EVALUATED:
200/metaphases/dose


DETERMINATION OF CYTOTOXICITY
- Method:


-Scoring of Chromosome Damage:
In each slide, 100 metaphases (200 metaphases/dose) were examined using the biological microscope of differential interference type (BX51, Olympus, Japan) of 1 ODD-fold magnification. Structural aberration was classified as follows; chromatid break (ctb), chromatid exchange (cte), chromosome break (csb), chromosome exchange (cse) and other (0). Numerical aberration was classified as follows polyploidy (pol). Two types aberration such as chromatid and chromosome gap (g) were recorded separately. In the meta phase, if there are several gap or cuttings, it was recorded as a fragment (frg). For a numerical aberration, any cell with 1 or more aberration was counted as 1 aberrant cell. Evaluation of results did not include gaps while the gaps were recorded in raw data of structural aberration. For numerical aberration, any cell with 1 or more polyploidy (pol) aberration was counted as 1 aberrant cell.

OTHER EXAMINATIONS:
- Determination of polyploidy:
Frequency of polyploid cells

Evaluation criteria:

The final decision of chromosome aberration in relation to the test substance was carried out in accordance with Toshio Sofuni and et al. [4]. If an an appearance rate was below 5% between 5 and 10% and over 10%, it was judged as a negative, equivocal, and positive, respectively

Statistics:

Statistical analysis was not performed. The mean value and standard deviation were calculated using the values measured from the test.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Refer to information on results and attached tables.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Range finding studies:
Growth inhibition test (Table 1)
A growth inhibition test was conducted in 9 dose levels, in which the highest dose of 5000 µg/mL is sequentially diluted to 8 additional lower doses (5, 10, 50, 100,250,500, 1000,2500 µg/mL) with the negative control groups. As the result of the growth inhibition test, the ICso was calculated as 9.2 µg/mL and 422.1 µg/mL for the short time treatment without and with metabolic activation system and 8.9 µg/mL for the continuous treatment.

2. In vitro chromosome aberration test (Table 2, 3)
On the basis of the growth inhibition test, the highest dose in chromosome aberration test was chosen as 10 µg/mL for the short time treatment without metabolic activation system and the continuous treatment, and 420 µg/mL for the short time treatment with metabolic activation system. Subsequently, the highest dose formulations were diluted by common ratio of 2 to produce 2 additional lower doses levels accompanied by a negative and positive control. Each dosing formulations were treated for 6 and 24 hours for the short time treatment regardless of metabolic activation and continuous treatment without metabolic activation, respectively; and specimens were prepared to examine chromosome aberration.

As a result, the structural and numerical chromosome aberrations were not observed in both the short time treatment with and without metabolic activation system and the continuous treatment as the negative control.

The cell proliferation rate in 0, 2.5, 5 and 10 µg/mL were calculated as 100.0, 102.7, 98.6 and 98.6 % for short time treatment without metabolic activation system, and in 0, 105, 210, 420 µg/mL were calculated as 100.0, 115.0, 115.0 and 82.5 % for short time treatment with metabolic activation system. In addition, the cell proliferation rate in 0, 2.5,5,10 µg/mL were calculated as 100.0,101.3,117.7, 72.2 % for continuous treatment system.

According to the chromosomal aberration test, the result was negative for the short time treatment with metabolic activation. In order to verify the result, the confirmation test was carry out in 6 hours after the cultivation, recovery time executed for 42 hours.

Result of confirmation test, the number of structural and numerical chromosome aberration was not increased as compared with that of a negative control.

This study was designed to examine the clastogenic potential of 2-(2-Vinyloxyethoxy) ethyl acrylate in the chromosome aberration test system using Chinese hamster lung cell.

The result of growth inhibition test showed that the IC50 was calculated as 9.2 µg/mL and 422.1 µg/mL for the short time treatment without and with metabolic activation system and 8.9 µg/mL for the continuous treatment. On the basis of the growth inhibition test, the highest dose in chromosome aberration test was chosen as 10 µg/mL for the short time treatment without metabolic activation system and the continuous treatment and 420 µg/mL for the short time treatment with metabolic activation system. Subsequently, the highest dose formulations were diluted by common ratio of 2 to produce 2 additional lower doses levels accompanied by a negative and positive control.

The structural and numerical chromosome aberrations were not observed in both the short time treatment with and without metabolic activation system and the continuous treatment as the negative control.

Form that the result showed negative for the short time treatment with metabolic activation, the confirmation test was performed to verify the negative result by 6 hours treatment and 42 hours recovery.

Through the confirmation test, it was also found that the number of structural and numerical chromosome aberration for the short time treatment with metabolic activation system was not increased as compared with that of a negative control.

It is confirmed that averages of chromosome aberration cells in negative and positive control group were within the range of the historical control data in our laboratory (Table 4).

In conclusion, 2-(2-Vinyloxyethoxy) ethyl acrylate did not show the chromosome aberrations regardless of application of metabolic activation system in the chromosome aberration test system using Chinese hamster lung cell (CHL/IU), under the conditions of this study. -

Conclusions:

In conclusion, 2-(2-Vinyloxyethoxy) ethyl acrylate did not show the chromosome aberrations regardless of application of metabolic activation system in the chromosome aberration test system using Chinese hamster lung cell (CHL/IU), under the conditions of this study.

Executive summary:

This study was designed to examine a clastogenic potential of 2-(2-Vinyloxyethoxy)ethyl acrylate in the chromosome aberration test system using Chinese hamster lung cell (CHL/IU).

The result of growth inhibition test showed that the IC50 (Inhibition concentration 50%) was calculated as 9.2 µg/mL and 422.1 µg/mL for the short time treatment without and with metabolic activation system and 8.9 µg/mL for the continuous treatment. On the basis of the growth inhibition test, the highest dose in chromosome aberration test was chosen as 1 0 µg/mL for the short time treatment without metabolic activation system and the continuous treatment, and 420 µg/mL for the short time treatment with metabolic activation system. Subsequently, the highest dose formulations were diluted by common ratio of 2 to produce 2 additional lower doses levels accompanied by a negative and positive control.

Each dosing formulations were treated for 6 and 24 hours for the short time treatment regardless of metabolic activation and continuous treatm~nt without metabolic activation, respectively; and specimens were prepared to examine chromosome aberration.

As the result of chromosome aberration test, the number of structural and numerical chromosome aberration cells of treatment groups was not increased as compared with that of a negative control group regardless of application of metabolic activation system in the short time treatment and continuous treatment.

From that the result showed negative for the short time treatment with metabolic activation, the confirmation test was performed to verify the negative result by 6 hours treatment and 42 hours recovery.

Through the confirmation test, it was also found that the number of structural and numerical chromosome aberration for the short time treatment with metabolic activation system was not increased as compared with that of a negative control. It is confirmed that averages of chromosome aberration cells in negative and positive control group were within the range of the historical control data in the laboratory.

In conclusion, 2-(2-Vinyloxyethoxy)ethyl acrylate did not show the chromosome aberrations regardless of application of metabolic activation system in the chromosome aberration test system using Chinese hamster lung cell (CHL/IU), under the conditions of this study.

Reference 3

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:

Between 5 March 2002 and 8 May 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations form standard test guidelines and/or minor methodlogical deficiencies, which do not affect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

other: This study was conducted as per the revised procedures that require for Testing New Chemical Substances (Kampoan No. 287, Eisei No. 127: October 31, 1997; Kikiyoku No.2: October 31, 1997.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of signature: 05/05/2002 Date of inspection: Not stated.

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine locus: Salmonella typhimurium
Tryptophane locus: Escherichia coli

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital and 5,6-Benzoflavone induced rat liver S9 mix.

Test concentrations with justification for top dose:

Dose selection test:
5, 10, 50, 100, 500, 1000 and 5000 µg/plate.

Mutagenicity test:
With metabolic activation: 312.5, 625, 1250, 2500 and 5000 µg/plate
Without metabolic activation: 156.3, 312.6, 625, 1250, 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:
DMSO

- Justification for choice of solvent/vehicle:
According to the sponsor, the test subtance was insoluble in water, but soluble and stable in DMSO. Therefore, the solvent, DMSO was selected for the test.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4 positive control substances were used: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide; Sodium azide; 9-Aminoacridine and 2-Aminoanthracene.

Details on test system and experimental conditions:

METHOD OF APPLICATION:
A 0.1 mL of each concentration of test substance or 0.1 mL of negative or positive control, 0.5 mL of S9 mix (with metabolic activation) or 0.1 mol/L phosphate buffer, pH 7.4 (without metabolic activation) and 0.1 mL of each tester strain were taken into heat sterilized glass tube (13 x 100 mm), mixed and incubated for 20 min at 37 deg C with agitation in a water bath shaken (SHK-100B, IWAKI GLASS). Then 2mL of molten top agar of 45 deg C was added to each tube, mixed and plated by pouring into pre-numbered minimal glucose agar (AMT-O) plate. Duplicate plates were used for each dose in dose determined and mutagenicity tests except for the negative control of mutagenicity test in which triplicate plates were used.

DURATION
- Preincubation period:
10 hours

- Exposure duration:
48 hours.

- Expression time (cells in growth medium):
48 hours.

- Selection time (if incubation with a selection agent):
Not applicable.

- Fixation time (start of exposure up to fixation or harvest of cells):
48 hours.

SELECTION AGENT (mutation assays):
Not applicable.

SPINDLE INHIBITOR (cytogenetic assays):
Not applicable.

STAIN (for cytogenetic assays):
Not applicable

NUMBER OF REPLICATIONS:
2 in test doses, 3 in negative control doses.

NUMBER OF CELLS EVALUATED:
Not applicable.

DETERMINATION OF CYTOTOXICITY
- Method:
Colony count: After completion of incubation, the colonies of all plates except for positive control plates were counted manually. Each of the positive control plate was counted three times using an auto colony counter and average of such counts was expressed as revertant colonies per plate. The mean number of colonies in duplicate or triplicate plates was expressed as revertant colonies per dose.

Observation of background loan:
During colony count, bactericidal effect of the test substance (bacterial growth inibition) was determined from the background lawn under stereozoom microscope (CSZ, Uchida-Yoko Ltd) and at the same time precipitates of the test substance was also determined.

OTHER EXAMINATIONS:
None stated.

Evaluation criteria:

The test substance was judged positive (+) when the number of revertant colonies in the test substance treated plates increased dose dependently and became 2-fold compared to that of the negative control and this effect was reasonably reproducible. The others were judged negative (-), Bactericidal effect (bactericidal growth inhibition) was judged when the lawn of the test plate was sparse or thin compared to that of the negative control plate.

Statistics:

The statistical analysis was not done for the judgement.

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000µg/plate without metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000µg/plate without metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000µg/plate without metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000µg/plate without metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000µg/plate without metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:
Not stated.

- Effects of osmolality:
Not stated.

- Evaporation from medium:
Not stated.

- Water solubility:
Not stated.

- Precipitation:
No precipitation noted

- Other confounding effects:
Not staed.

RANGE-FINDING/SCREENING STUDIES:
A dose selection test was conducted at the doses of 5, 10, 50, 100, 500, 1000 and 5000 µg/plate either in the presence or absence of metabolic activation system to find out a dose at which the test substance inhibited bacterial growth and caused precipitations.

COMPARISON WITH HISTORICAL CONTROL DATA:
The numbers of revertant colonies in the negative and positive controls of all test strains were similar to the mean ± 2SD of the background data (attached sheet 1) which demonstrated that the study was conducted appropriately.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Not stated.

A dose selection test was conducted at the doses of 5, 10, 50, 100, 500, 1000 and 5000 µg/plate either in the presence or absence of metabolic activation system to find out a dose at which the test substance inhibited bacterial growth and caused precipitations. In the test, bactericidal effect (bacterial growth inhibition) was noted at the dose of 5000 µg/plate in all tester strains in the absene of metabolic activation system. But, precipitations of the test substance were not noted. Further, the numbers of revertant colonies in the test substance treated plates were not increased (Appendix 1 in attachment 2).

Based on such results, the dose of 5000µg/plate that caused bactericidal effect considered as highest and lower doses of 2500, 1250, 625, 312.6 and 156.3 µg/plate at a nominal ration of 2 (total 6 doses) were selected and used in mutagenicity test in the absence of metabolic activation system. Whereas, the doses of 5000 (highest), 2500, 1250, 625 and 312.5 µg/plate at a nominal ratio of 2 (total 5 doses) were selected and used in mutagenicity test in the presence of metabolic activation system.

As a result, the number of revertant colonies in the test substance treated plates of all tester strains were not increased dose dependently and not became 2-fold compared to that of the negative control of each tester strain. Further, bactericidal effect was noted at the dose of 5000 µg/plate in all tester strains in the absence of metabolic activation system. But, precipitations of the test substance were not noted (see Appendix 2 in attachment 2).

Positive controls of all tester strains showed marked increase in the number of revertant colonies compared to that of the corresponding negative control of each tester strain.

Conclusions:

According to the findings of this experimental condition HBM-AV was concluded as non-mutagen.

Executive summary:

The mutagenicity of HBM-AV was examined using histadine requiring tester strains of TA98, TA100, TA1535 and TA1537 (Salmonella typhimurium) and tryptophane requiring strain of WP2uvrA (Escherichia coli) either in the presence or absence of metabolic activation system.

The tested doses of 156.3, 312.5, 625, 1250, 2500, 5000 µg/plate were used in the absence of metabolic activation system. Whereas, the doses of 312.5, 625, 1250, 2500 and 5000 µg/plate were used the presence of metabolic activation system. As a result, the numbers of revertant colonies in the test substance treated plates of the tester strains were not increased at least twice the concurrent negative controls in a dose-dependant manner and the findings were rather reproducible due to similar result in the dose selection test. Further, bacterial effect of the test substance was noted at the dose of 5000 µg/plate in the tester strains in the absence of the metabolic activation system. But, precipitations of the test substance were not noted. Furthermore, the numbers of revertant colonies of the positive controls of all tester strains were remarkably increased compared to their respective negative controls.

The findings concluded that HBM-AV had no mutagenicity under this experimental condition.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The experimental phases of the study were performed between 12 May 2008 and 07 July 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of therelevant results.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 19/08/08 Date of signature: 04/03/09

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media:
RPMI 1640

- Properly maintained:
yes

- Periodically checked for Mycoplasma contamination:
yes

- Periodically checked for karyotype stability:
no

- Periodically "cleansed" against high spontaneous background:
yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and beta-naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

Experiment 1: 2.5 to 60 µg/ml in the absence of metabolic activation and 27.5 to 460 µg/ml in the presence of metabolic activation.
Experiment 2: 10 to 60 µg/ml in the absence of metabolic activation and 27.5 to 460 µg/ml in the presence of metabolic activation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:
arachis oil BP

lot number - W72

storage conditions - Room temperature

date recieved - 05/02/2008

- Justification for choice of solvent/vehicle:
Not stated

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Solvent (R0 medium) treatment groups were used as the vehicle controls.

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation Migrated to IUCLID6: lot number A0164185

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Solvent (R0 medium) treatment groups were used as the vehicle controls.

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION:

DURATION
- Preincubation period: not applicable
- Exposure duration: not applicable
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 10 to 14 days
- Fixation time (start of exposure up to fixation or harvest of cells): 12 to 16 days

SELECTION AGENT (mutation assays): 5TFT
SPINDLE INHIBITOR (cytogenetic assays): Cyto B
STAIN (for cytogenetic assays): Giemsca

NUMBER OF REPLICATIONS:
duplicate

NUMBER OF CELLS EVALUATED:
96 - (wells) x 2000 per plate (4 plates per dose level)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
-colony size

Statistics:

All data were statistically anaylsed using appropriate statistical methods as recommended by the UKEMS Sub-committee on Guidelines for Mutagenicity Testing Report, Part III (1989). The data was analysed following a √(x +1) transformation using the student;s t-test (two tailed) and any significant results were confirmed using the one way analysis of variance.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

non-mutagenic

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: not stated
- Effects of osmolality: not stated
- Evaporation from medium: not stated
- Water solubility: not stated
- Precipitation: not stated
- Other confounding effects: not stated


RANGE-FINDING/SCREENING STUDIES:
See any other information on results section

COMPARISON WITH HISTORICAL CONTROL DATA:


ADDITIONAL INFORMATION ON CYTOTOXICITY:
Experiment 1
The results of the microtitre plate counts and their analysis are presented in Tables 2 to 7.
There was evidence of toxicity following exposure to the test material in both the absence and presence of metabolic activation, as indicated by the %RSG and RTG values. There was evidence of modest reductions in (%V) viabilities in both the absence and presence of metabolic activation, indicating that some residual toxicity had occurred. Optimum levels of toxicity were achieved in both the absence and presence of metabolic activation. The toxicity observed at 60 µg/ml in the absence of metabolic activation and at 460µg/ml in the presence of metabolic activation exceeded the upper acceptable limit of 90%, therefore, this dose was excluded from the statistical analysis. Acceptable levels of toxicity were seen with both positive control substances (Table 3 and Table 6).
Neither of the vehicle control mutant frequency values were outside the range of 50 to 200 x 10-6 viable cells that is acceptable for L5178Y cells at Harlan Laboratories Ltd. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (Tables 3 and 6).

The test material induced modest but statistically significant dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell in both the absence and presence of metabolic activation (Table 3). In the presence of metabolic activation, a statistically significant increase in mutant frequency was observed at 400 µg/ml. However, the increase in mutant frequency observed at this dose level did not exceed the GEF value, was at the limit of acceptable toxicity, and the mutant frequency value observed was within the acceptable range for vehicle controls. Therefore, the response observed was considered to be of no toxicological significance (Table 6). However in the absence of metabolic activation, statistically significant increases in mutant frequency were observed at and above 40 µg/ml. The increase in mutant frequency observed at 50 µg/ml exceeded the GEF value and was also higher than the current historical range for vehicle controls. However, the increase in mutant frequency that exceeded GEF was once again seen at a dose level at the limit of acceptable toxicity and, therefore, may have been due to a cytoxic mechanism rather than a true genotoxic response (Table 3). Therefore, the exposure period in the absence of metabolic activation in Experiment 2 was not extended to 24 hours and remained at 4-hours to confirm or disprove any reproducibility of the response observed. No precipitate of the test was observed at any dose levels.


The numbers of small and large colonies and their analysis are presented in Tables 4 and 7.

Experiment 2
As was seen in experiment 1 there was no evidence of toxicity following exposure to the test material in both the absence and presence of metabolic activation, as indicated by the % RSG and RTG values. There was also once again evidence of modest reductions in (%V) viabilities in both the absence of and presence of metabolic activation, indicating that some residual toxicity ad occurred. Optimum levels of toxicity were achieved in both the absence and presence of metabolic activation. The excessive levels of toxicity observed at and above 400µg/ml in the presence of metabolic activation resulted in these dose levels not being plated for viability and TFT resistance. The toxicity observed at 60 µg/ml in the absence of metabolic activation and at 340 µg/ml in the presence of metabolic activation exceeded the upper acceptable limit of 90%, theerfore, these dose levels were excluded from the statistical analysis. Acceptable levels of toxicity were seen with both positive control substances (Tables 9 and 12).

The lowering of the S9 concentration to 1% S9 in this second experiment resulted in greater levels of toxicity than those observed in the presence of 2% S9 in the first experiment.

Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 x 10E -6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (Tables 9 and 12).

The test material once again induced statistically significant dose related (linear trend) increases in the mutant frequency in both the absence and the presence of metabolic activation. On this occasion the GEF was not exceeded in the absence of metabolic activation but exceeded in the presence of metabolic activation. The increases in mutant frequency observed at the upper surviving dose levels, were once again at the limit of acceptable toxicity in both the absence and presence of metabolic activation, and were only marginally higher than the acceptable range for vehicle controls. There were no reproducible increases in mutant frequency that exceeded the GEF value between the two experiments, in either the absence or presence of metabolic activation. The responses observed were considered to be due to a cytotoxic mechanism and not a true genotoxic response and, therfore, of no toxicological significance (tables 9 and 12). No precipitate of test material was observed at any of the dose levels.

The numbers of small and large colonies and their analysis are presented in Tables 10 and 13.

Preliminary Toxicity Test

The dose range of the test material used in the preliminary toxicity test was 7.27 to 1862 µg/ml. The results for the Relative Suspension Growth (%) were as follows:

Dose (µg/ml)	%(-S9) 4-Hour Exposure	%(+S9) 4-Hour Exposure	%(-S9) 24-Hour Exposure
0	100	100	100
7.27	83	120	71
14.55	69	110	35
29.09	54	100	8
58.19	9	95	0
116.38	1	95	0
232.75	0	78	0
465.5	0	8	0
931	0	1	0
1862	0	0	0

In all three of the exposure groups there was a marked reduction in the Relative Suspension Growth (%RSG) of cells treated with the test material when compared to the concurrent vehicle controls. The toxicity curve was steep in all three exposure groups. Precipitate of the test material was not observed at the end of the exposure periods. In the subsequent mutagenicity experiments the maximum dose was limited by toxicity.

Conclusions:

The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

Executive summary:

Introduction:

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method used meets the requirements of the OECD (476) of Commision Regulation (EC) No. 440/2208 of 30 May 2008.

Methods:

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 the test material 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, the exposure period in the absence of metabolic activation would normally be extended to 24 -hours. However, due to the small but statistically significant response observed in the absence of metabolic activation in the first experiment, and also the very high levels of toxicity observed in the 24 -hour exposure group of the Preliminary Toxicity Test, it was considered inappropriateto investigate the 24 -hour exposure period. Therefore, in the second experiment the cells were treated with the test material at eight dose levels using 4 -hour exposure groups both in the absence and presence of metabolic activation (1% S9).

The dose range of the test material was selected following the results of a prelimiary toxicity test and for the first experiment were 2.5 to 60 µg/ml in the absence of metabolic activation and 27.5 to 460 µg/ml in the presence of metabolic activation. For the second experiment the dose range was 10 to 60 µg/ml in the absence of metabolic activation and 27.5 to 460 µg/ml in the presence of metabolic activation.

Results:

The maximum dose level used was limited by test material induced toxicity. Precipitate of test material was not observed at any of the dose levels in the mutagenicity test. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for hte L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material induced very modest but statisticallly significant dose-related increases in the mutant frequency in the absence and presence of metabolic activation, in both the first and second experiment. However, it should be noted that the responses which exceeded the GEF value were at dose levels where the toxicity was at the very limit of acceptability, were not reproducible between the two experiments and with the exception of the 4 -hour exposure group in the absence of metabolic activation in the first experiment the mutant frequency values observed were within or marginally higher than the acceptable range for vehicle controls. It was therefore considered that responses observed were due to a cytotoxic mechanism rather than a true genotoxic response and were, therefore, considered to have no toxicological significance.

Conclusion:

The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The following studies are  available, all of which gave negative results.

- two in-vivo mouse micronucleus studies

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

between 21 January 2009 and 13 March 2009.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations form standard test guidelines and/or minor methodlogical deficiencies, which do not affect the quality of the relevant results.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 19/08/08 Date of signature: 04/03/09

Type of assay:

micronucleus assay

Species:

mouse

Strain:

other: albino Hsd: ICR (CD-1®)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan, UK
- Age at study initiation: five to eight weeks old.
- Weight at study initiation: 24 to 30g
- Housing: The animals were housed in groups of up to seven, by sex, in solid-floor polypropylene
cages with wood-flake bedding.
- Diet (e.g. ad libitum): Free access to food (Harlan Teklad 2014 Rodent Pelleted Diet) was allowed throughout the study.
- Water (e.g. ad libitum): Free access to mains drinking water was allowed throughout the study.
- Acclimation period: After a minimum acclimatisation period of five days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25°C
- Humidity (%): 30 to 70% respectively
- Air changes (per hr): The rate of air exchange was approximately fifteen changes per hour
- Photoperiod (hrs dark / hrs light): the lighting was controlled by a time switch to give twelve hours light and twelve hours darkness.


IN-LIFE DATES: From: Day 0 To: End of study

Route of administration:

oral: gavage

Vehicle:

The vehicle was supplied by Lab 3 Ltd, as follows:

Supplier's identification: Arachis Oil BP
Supplier's lot number: W72
Date received: 05 February 2008
Description: Pale straw coloured slightly viscous liquid
Storage conditions: Room temperature

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test material was freshly prepared as required as a solution at the appropriate concentration in arachis oil.

DIET PREPARATION:
Not applicable.

Duration of treatment / exposure:

One group of mice from each dose level was killed by cervical dislocation 24 hours following treatment and a second group dosed with test material at 1500 mg/kg was killed after 48 hours.

The vehicle controls were killed 24 or 48 hours following dosing and positive control group animals were killed 24 hours following dosing.

Frequency of treatment:

Mice dosed once only.

Post exposure period:

All animals were observed for signs of overt toxicity and death one hour after dosing and then once daily as applicable and immediately prior to termination.

Dose / conc.:

1 500 mg/kg bw/day (nominal)

Dose / conc.:

750 mg/kg bw/day (nominal)

Dose / conc.:

375 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Groups, each of seven mice, were dosed once only via the oral route with the test material at 1500, 750 or 375 mg/kg.

In addition, three further groups of mice were included in the test; two groups (each of seven mice) were dosed via the oral route with the vehicle alone (arachis oil) and a third group (five mice) was dosed orally with cyclophosphamide.

Control animals:

yes, concurrent vehicle

Positive control(s):

Supplier's identification: Cyclophosphamide
Supplier's lot number: A0164185
Date received: 30 October 2008
Storage conditions: Approximately 4°C in the dark
PROJECT NUMBER: 0706/0150

For the purpose of this study the positive control material was freshly prepared as required as a solution at the appropriate concentration in distilled water.

- Justification for choice of positive control(s): Cyclophosphamide is a positive control material known to produce micronuclei under the conditions of the test.

Tissues and cell types examined:

Stained bone marrow smears were coded and examined blind using light microscopy at x1000 magnification. The incidence of micronucleated cells per 2000 polychromatic erythrocytes (pCE-blue stained immature cells) per animal was scored. Micronuclei are normally circular in shape, although occasionally they may be oval or half-moon shaped, and have a sharp contour with even staining. In addition, the number of normochromatic
erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes was counted; these cells were also scored for incidence of micronuclei.
The ratio of polychromatic to normochromatic erythrocytes was calculated together with appropriate group mean values and standard deviations.

Details of tissue and slide preparation:

Slide preparation:
Immediately following termination (i.e. 24 or 48 hours following dosing), both femurs were dissected from each animal, aspirated with foetal calf serum and bone marrow smears prepared following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol, stained in May-Grilnwald/Giemsa, allowed to air-dry and cover-slipped using mounting medium.

Evaluation criteria:

Interpretation of results:
A comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the test material groups and the number occurring in the corresponding vehicle control group.

A positive mutagenic response was demonstrated when a statistically significant, doseresponsive, toxicologically relevant increase in the number of micronucleated polychromatic erythrocytes was observed for either the 24 or 48-hour kill times when compared to their corresponding control group.

If these criteria were not fulfilled, then the test material was considered to be non-genotoxic under the conditions of the test.

A positive response for bone marrow toxicity was demonstrated when the dose group mean polychromatic to normochromatic ratio was shown to be statistically significantly lower than the concurrent vehicle control group.

Statistics:

All data were statistically analysed using appropriate statistical methods as recommended by the UKEMS Sub-committee on Guidelines for Mutagenicity Testing Report, Part III (1989). The data was analysed following a √(x + 1) transformation using Student's t-test (two tailed) and any significant results were confirmed using the one way analysis of variance.

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Hunched posture and ptosis.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 1000 to 2000 mg/kg
- Clinical signs of toxicity in test animals: clinical signs were observed above 1500 mg/kg and included:
Hunched posture, ptosis, prostration, decreased respiration, laboured respiration and hypothermia.

The test material showed no marked difference in its toxicity to male or female mice; it was therefore considered to be acceptable to use males only for the main test. Adequate evidence of test material toxicity was demonstrated via the oral route of administration; therefore, this was selected for use in the main test. The maximum tolerated dose (MTO) of the test material, 1500 mg/kg, was selected for use in the main test, with 750 and 375 mg/kg as the lower dose levels.

RESULTS OF DEFINITIVE STUDY
Mortality Data and Clinical Observations:
There were no premature deaths seen in any of the dose groups. Clinical signs were observed in animals dosed with the test material at and above 750 mg/kg in both the 24 and 48-hour groups where applicable, these were as follows: Hunched posture and ptosis.

Evaluation of Bone Marrow Slides:
A summary of the results of the micronucleus test is given in Table 1. Individual and group mean data are presented in Tables 2 to 8 (see attached background material for tables).
A modest but statistically significant decrease in the PCE/NCE ratio was observed in the 24-hour 375 mg/kg test material group when compared to the concurrent vehicle control groups. However, with no evidence of any statistically significant decreases in the PCE/NCE ratio in any of the higher test material dose groups, the response was considered to be spurious and of no biological relevance. The observation of clinical signs, at and above 750 mg/kg in both the 24 and 48-hour groups where applicable, was taken to indicate that systemic absorption had occurred and exposure to the target tissue had been achieved.

There were no statistically significant increases in the frequency of micronucleated PCE in any of the test material dose groups when compared to their concurrent vehicle control groups.

The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.

The test material was found not to produce a significant increase in the frequency of micronuclei in polychromatic erythrocytes of mice under the conditions of the test.

Conclusions:

The test material was considered to be non-genotoxic under the conditions of the test.

Executive summary:

Introduction:

The study was performed to assess the potential of the test material to produce damage to chromosomes or aneuploidy when administered to mice. The method was designed to comply with the 1997 DECD Guidelines for Testing of Chemicals No.474 "Micronucleus Test", Method 812 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the USA EPA, TSCA and FIFRA guidelines and the Japanese METI/MHLW guidelines for testing of new chemical substances.

Methods:

A range-finding test was performed to find suitable dose levels of the test material, route of administration and to investigate to see if there was a marked difference in toxic response between the sexes. There was no marked difference in toxicity of the test material between the sexes; therefore the main test was performed using only male mice. The micronucleus test was conducted using the oral route in groups of seven mice (males) at the maximum tolerated dose (MTD) 1500 mg/kg and with 750 and 375 mg/kg as the two lower dose levels. Animals were killed 24 or 48 hours later, the bone marrow extracted, and smear preparations made and stained. Polychromatic (PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei.

Further groups, each of 7 mice, were given a single oral dose of arachis oil or dosed orally with cyclophosphamide (5 mice), to serve as vehicle and positive controls respectively. Vehicle control animals were killed 24 or 48 hours later, and positive control animals were killed after 24 hours.

Results:

There were no premature deaths seen in any of the dose groups. Clinical signs were observed in animals dosed with the test material at and above 750 mg/kg in both the 24 and 48-hour groups where applicable, these were as follows: Hunched posture and ptosis.

A modest but statistically significant decrease in the PCE/NCE ratio was observed in the 24-hour 375 mg/kg test material group when compared to the concurrent vehicle control group. However, with no evidence of any statistically significant decreases in the PCE/NCE ratio in any of the higher test material dose groups, the response was considered to be spurious and of no biological relevance. The observation of clinical signs, at and above 750 mg/kg in both the 24 and 48-hour groups where applicable, was taken to indicate that systemic absorption had occurred and exposure to the target tissue had been achieved.

There was no evidence of a significant increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test material when compared to the concurrent vehicle control groups.

The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.

Conclusion: The test material was considered to be non-genotoxic under the conditions of the test.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

from 2011-12-13 to 2012-04-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline-conform study performed under GLP without deviations

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: On the Test Method Concerning New Chemical Substances, etc. a confederative notification No. 0331-7 of the PFSB, MHLW, March 31, 2011; No. 5 of the Manufacturing Industries Bureau, METI, March 29, 2011;No. 110331009 of the Environmental Policy Bureau, MOE

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

other: Crlj:CD1 (ICR)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc.
- Age at study initiation: 7 weeks
- Weight at study initiation: males: 27.8 - 30.0 g, females: 22.0 - 26.1 g (preliminary test), 19.6 - 23.5 (main test)
- Assigned to test groups randomly: stratified random sampling based on the body weights on the day of group assignment so that mean body weights might be comparable among groups
- Fasting period before study: no
- Housing: in metal bracked cages with wire mesh floors in groups of 5 during quarantine and acclimatization and in groups of 1 or 2 during test
- Diet (e.g. ad libitum): free access to pellet diet
- Water (e.g. ad libitum): Sapporo City tap water was micro-filtrated and given to the animals ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): 10 -15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
Preliminary test: administration on 27 and 28 December 2011
Main test: administration on 17 and 18 January 2012

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: sesame oil
- Justification for choice of solvent/vehicle: Because of difficulty in preparation due to low water solubility of the test substance, sesame oil, an oil-soluble solvent, was used. Sesame oil is generally used as the vehicle to prepare a chemical substance which is hardly soluble in water for intraperitoneal administration in the test facility.
- Concentration of test material in vehicle: Preliminary test: 25, 50, 100, 200 mg/mL. Main test: 41.0, 51.2, 64, 80 mg/mL

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance was accurately weighed, to which the vehicle (sesame oil) was added and the test substance was dissolved to prepare the test solution of the highest concentration. The test solution of the highest concentration was serially diluted to prepare the test solutions of lower concentrations. The test solutions were prepared before use and used within 1.7 h after preparation in the preliminary test and within 1.5 h in the main test.

Duration of treatment / exposure:

Administered twice within approximately 24-h interval.

Frequency of treatment:

Administered twice within approximately 24-h interval.
The dosing frequency and interval were selected because preparing specimens 18 to 24 h after the final dose allows examination of micronuclei 18 to 48 h after dosing; which is likely to enable evaluation of the potential of the test substance to induce micronuclei.

Post exposure period:

Observation immediately before each dose, until approximately 6 h after each dose, as well as 22 to 24 h (preliminary test) or 21 to 23 h (main test) after the final dose.
Weighing before each dose, and 22 to 23 h (preliminary test) or 21 to 23 h (main test) after the final dose

Remarks:

Doses / Concentrations:
250, 500, 1000, 2000 mg/kg/day
Basis:
nominal conc.
(Preliminary test)

Remarks:

Doses / Concentrations:
410, 512, 640, 800 mg/kg/day
Basis:
nominal conc.
(Main test)

No. of animals per sex per dose:

Preliminary test: 3 males and 3 females per dose group
Main test: 6 females per treatment group, 6 females per reference control group, 6 females in positive control group

Control animals:

yes, concurrent vehicle

Positive control(s):

Positive Control: mitomycin C
- Justification for choice of positive control(s): not reported, but suggested in OECD TG 474
- Route of administration: intraperitoneal
- Vehicle: Japanese Pharmacopoeia water
- Volume administered: 10 mL/kg/time
- Doses / concentrations: 1 mg/kg/day
- Frequency: The positive control substance was administered once, which is the common dosing frequency of the positive control substance in this test method.

Reference Control: Ethyl acrylate
- Justification for choice of positive control(s): not reported
- Route of administration: intraperitoneal
- Vehicle: sesame oil
- Volume administered: 10 mL/kg/time
- Doses / concentrations: 600, 800, 1000 mg/kg/day
- Other: The reference control substance was administered once because two-time administration causes severe toxicity that makes evaluation at the high dose difficult.

Tissues and cell types examined:

- bone marrow cells: prepared 23 to 24 h after final dose

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
In the preliminary test, all 3 females in the 2000 mg/kg/day group were found dead on the day after the first dose, and all 3 males in this group died before euthanasia on the day after the first dose. In the 1000 mg/kg/day group, 2 of 3 males and females were found dead on the day after the final dose. No abnormalities in general appearance or deaths were noted in males or females in the 250 or 500 mg/kg/day group. Although no differences were noted in the number of males and females that died, deaths of females in the 2000 mg/kg/day group tended to occur earlier. Accordingly, females were selected in the main test. The LD50 values of the test substance were estimated to be around 900 mg/kg/day in males and females; therefore, the highest dose was set at 800 mg/kg/day, the estimated maximum tolerable dose, and 3 lower doses were set in a serial dilution at 1.25-fold dilution series (4 doses in total).

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): no additional information necessary

DETAILS OF SLIDE PREPARATION:
Preparation of the animals
- speciments prepared from 5 surviving animals in each test group
- euthanization by cervical dislocation
- bone marrow cells in the right and left femurs flushed with fetal bovine serum
- cell suspension centrifuged at 1000 rpm (150 x g) for 5 minutes to remove excess serum
- part of resultant cell suspension smeared onto a slide glass
- air drying of the smear and fixing with methanol
- four specimens prepared from each animal
- two of the four specimens blind coded and stained with 0.005 % acridine prange stain
- washed with 1/15 mol/L phosphate buffer, covered wiht glass and sealed wiht enamel

METHOD OF ANALYSIS:
- specimens observed with fluorescence microscope
- per animal 2000 immature erythrocytes observed and incidence of micronucleated immature erythocytes was calculated
- determination of the ratio between immature erythrocytes and total erythocytes based on observation of 500 erythocytes per animal

Evaluation criteria:

The validity of the assay was determined by the mean incidences of micronuclei in the negative and positive control groups are within the range of mean ± 3 SD calculated from the respective historical control data. Although a proportion of immature erythrocytes at any dose of the test substance or reference control that was less than 20% of the negative control group was to be excluded from evaluation, no data applied to this case.
The result was determined to be positive when the conditional binomial test showed that an incidence of micronuclei in a test substance group or reference
control group was significantly higher than in the negative control group in a dose-responsive manner.

Statistics:

Body weight: Bartlett’s test, Dunnett’s test (two-tailed), Steel’s test (two-tailed)
Incidence of micronuclei: conditional binomial test
Proportion of immature erythrocytes: Bartlett’s test, Dunnett’s test, Steel’s test, Cochran-Armitage trend test

Sex:

female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

640 and 800 mg/kg/day: abnormalities in general appearance, myelosuppression indicated by the decrease in the proportion of immature erythrocytes among total erythrocytes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 250 - 2000 mg/kg/day
- Solubility: Soluble in sesame oil
- Clinical signs of toxicity in test animals:
200 mg/kg/day: no abnormalities noted
500 mg/kg/day: no abnormatlities noted
1000 mg/kg/day: All 3 males showed staggering gait by 6 h after the first dose, and 1 male showed staggering gait and another 1 male showed a decrease in locomotor activity and hypothermia approximately 24 h after the first dose. Following the final dose, 1 male showed a decrease in locomotor activity, hypothermia, staggering gait, prone position, tachypnea, and tremor; 1 male showed a decrease in locomotor activity, and hypothermia; and 1 male showed a decrease in locomotor activity, hypothermia, prone position, tachypnea, and tremor by 6 h after the final dose. On the day after the final dose, 2 males were found dead, and 1 male showed a decrease in locomotor activity, hypothermia, and bradypnea. Two of 3 females showed a decrease in locomotor activity approximately 24 h after the first dose. Following the final dose, 1 of 3 females showed hypothermia, prone position, bradypnea, and tremor, and 1 female showed staggering gait, hypothermia, prone position, tachypnea, bradypnea, and tremor by 6 h after the final dose. On the day after the final dose, 2 females were found dead, and the other female showed abdominal distention.
2000 mg/kg/day: All 3 males showed a decrease in locomotor activity, hypothermia, bradypnea, staggering gait, prone position, and tachypnea, and 1 male showed also
lateral position by 6 h after the first dose. On the day after dosing, all of these animals showed a decrease in locomotor activity, 2 males showed hypothermia, and 1 male showed bradypnea. Accordingly, final dose was not given to these animals from the stand point of animal protection and they were carried out of the animal room for the purpose of euthanasia, but they died before euthanasia. All 3 females showed a decrease in locomotor activity, hypothermia, bradypnea, staggering gait, prone position, and tachypnea by 6 h after the first dose. On the day after the first dose, all these females were found dead.
- Evidence of cytotoxicity in tissue analyzed: tissue not analyzed
- Rationale for exposure: As no information was obtained on the toxicity of the test substance administered to animals, the highest dose of the test substance was set at 2000 mg/kg/day, the upper limit specified in the test guideline, and 3 lower doses were set in a two-fold dilution series (4 doses in total).
- Harvest times: no harvest in preliminary test
- High dose with and without activation: not applicable

RESULTS OF DEFINITIVE STUDY
- General appearance: No abnormalities were noted in general appearance in the 410 or 512 mg/kg/day group. Abdominal hardening was noted in 5 of 6 animals after the final dose in the 640 mg/kg/day group. In the 800 mg/kg/day group, the following findings were noted after the final dose: 2 of 6 animals showed abdominal hardening; another 1 showed staggering gait and abdominal hardening; and another 1 showed a decrease in locomotor activity, hypothermia, staggering gait, and abdominal hardening, and was found dead on the following day.
- Induction of micronuclei (for Micronucleus assay): The incidences of micronuclei were 0.10% ± 0.04%, 0.17% ± 0.07%, 0.11% ± 0.08%, and 0.15% ± 0.09% in the 410, 512, 640, and 800 mg/kg/day groups, respectively, which were comparable to those in the negative control group, with no statistically significant differences.
- Ratio of PCE/NCE (for Micronucleus assay): The proportions of immature erythrocytes were 61.1% ± 4.7%, 61.5% ± 6.0%, 33.4% ± 15.6%, and 26.0% ± 17.6% in the 410, 512, 640, and 800 mg/kg/day groups, respectively. Those in the 640 and 800 mg/kg/day groups were statistically significantly lower than in the negative control group. In the decrease in the proportion of immature erythrocytes, dose-response relationship was detected by the Cochran-Armitage trend test.
- Appropriateness of dose levels and route: Abnormalities in general appearance were noted in the 640 and 800 mg/kg/day groups, and 1 animal in the 800 mg/kg/day group died. In both groups, myelosuppression was indicated by the decrease in the proportion of immature erythrocytes among total erythrocytes. Thus, sufficient exposure of animals to the test substance was considered to be achieved at the doses used in this assay.
- Statistical evaluation: The incidences of micronuclei were compared between the negative control group and each of the other groups (including the positive control group) using the conditional binomial test (Kastenbaum and Bowman’s table: Marvin A. Kastenbaum and K. O. Bowman. 1970. Tables for determining the statistical significance of mutation frequencies, Mutation Res., 9: 527-549). The test was performed at an upper significance level of 5%. No significant differences were noted by the conditional binomial test, and so further analysis was not performed.
The proportions of immature erythrocytes were analyzed by Bartlett’s test for homogeneity of variance between the negative control group and each test substance group or each reference control group. The results in the negative control group and test substance groups showing an evidence of homogeneity of variances (p > 0.05) were analyzed by Dunnett’s test, and those in the negative control group and reference control substance groups showing heterogeneity of variances (p ≤ 0.05) were analyzed by Steel’s test. Significance level was set at 5% (two-tailed) for comparative analysis with the negative control group, and p values were represented in the table separately at p ≤ 0.05 and p ≤ 0.01. Significant differences were noted in both analyses in the test substance groups and reference control groups; therefore, the proportions of immature erythrocytes were analyzed for dose-response by the Cochran-Armitage trend test. Significance level was set at 5% (two-tailed). The proportions of immature erythrocytes in the negative and positive control groups were analyzed for homogeneity of variances using the F test (two-tailed). The results showed homogeneity of variances (p > 0.05); therefore, Student’s t-test (two-tailed) was performed for comparison between the two groups at a significance level of 5%.

Results of the micronucleus assay with VEEA and ethyl acrylate in female mice

Compound	Dose^a [mg/kg/day or mg/kg]	Incidence of micronucleated immature erythrocyte (based on 2000 immature erythrocytes per animal) [%]	Proportion of immature erythrocyte (based on 500 erythrocytes per animal) [%]
Control (sesame oil)	-	0.15 ± 0.006 (n=5)	54.6 ± 9.1 (n=5)
VEEA	410	0.10 ± 0.04 (n=5)	61.1 ± 4.7 (n=5)
	512	0.17 ± 0.07 (n=5)	61.5 ± 6.0 (n=5)
	640	0.11 ± 0.08 (n=5)	33.4 ± 15.6 (n=5)^#
	800	0.15 ± 0.09 (n=5)	26.0 ± 17.6 (n=5)^##
Ethyl acrylate	600	0.13 ± 0.04 (n=5)	49.7 ± 6.3 (n=5)
	800	0.08 ± 0.06 (n=5)	29.7 ± 15.7 (n=5)
	1000	0.14 ± 0.07 (n=5)	35.9 ± 3.4 (n=5)^+
Mitomycin C	1	2.54 ± 0.54 (n=5) **	53.3 ± 5.3 (n=5)

^aTwo successive intraperitoneal injection (24 hours apart, VEEA) or single intraperitoneal injection (ethyl acrylate and mitomycin C)

^#and^##: Statistically significant difference form the control, p<=0.05 and 0.01, respectively (Dunnett’s test)

**Statistically significant difference from the control, p<= 0.01 (the Conditional binominal test)

⁺Statistically significant difference from the control, p<=0.05 (Steel’s test)

Conclusions:

The results indicated that 2-(2-Vinyloxyethoxy) ethyl acrylate did not induce micronuclei under the test conditions which ensured sufficient exposure of animals to the substance. 2-(2-Vinyloxyethoxy) ethyl acrylate was considered to be not clastogenic in vivo.

Executive summary:

In vivo clastogenic potential of 2-(2-Vinyloxyethoxy) ethyl acrylate (VEEA) was investigated by a micronucleus assay using bone marrow cells from mice intraperitoneally treated with VEEA. Ethyl acrylate as a reference control substance was also evaluated. Based on the results of the preliminary test using male and female mice, VEEA was administered to female mice at 410, 512, 640, and 800 mg/kg/day. As the results, the incidence of micronuclei did not increase in any test substance groups, which showed negative results. It is recommended in a micronucleus assay to select a dose that produces some signs of toxicity as the high dose level. In the present study, abnormalities in general appearance were noted in the 640 and 800 mg/kg/day groups, and 1 animal in the 800 mg/kg/day group died. In both groups, myelosuppression was indicated by the decrease in the proportion of immature erythrocytes among total erythrocytes. Thus, sufficient exposure of animals to the test substance was considered to be achieved at the doses used in this assay. Ethyl acrylate was administered to female mice at 600, 800, and 1000 mg/kg to investigate its potential to cause micronuclei. The results showed no increase in the incidence of micronuclei, which showed negative results and agreed with those in a previous report. Abnormalities in general appearance were noted in the 800 and 1000 mg/kg groups, and myelosuppression was indicated by the decreasing tendency or decrease in the proportion of immature erythrocytes among total erythrocytes. Thus, sufficient exposure of animals to the reference control substance was considered to be achieved at the doses used in this assay. The mean incidences of micronuclei in the negative and positive control groups were within the range of mean ± 3 SD calculated from the respective historical control data of the test facility. In addition, the incidence of micronuclei clearly increased in the positive control group, indicating that the test system had sufficient sensitivity, and that the potential of the test and reference control substances to induce micronuclei was appropriately evaluated. These results indicated that 2-(2-Vinyloxyethoxy) ethyl acrylate and the reference control substance, ethyl acrylate, did not induce micronuclei under the test conditions which ensured sufficient exposure of animals to these substances, and were considered to be not clastogenic in vivo.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In-vitro study results:

Bacterial reverse mutation assay (Ames study):

The mutagenicity of the test substance was examined using histadine requiring tester strains of TA98, TA100, TA1535 and TA1537 (Salmonella typhimurium) and tryptophane requiring strain of WP2uvrA (Escherichia coli) either in the presence or absence of metabolic activation system.

The tested doses of 156.3, 312.5, 625, 1250, 2500, 5000 µg/plate were used in the absence of metabolic activation system. Whereas, the doses of 312.5, 625, 1250, 2500 and 5000 µg/plate were used the presence of metabolic activation system. As a result, the numbers of revertant colonies in the test substance treated plates of the tester strains were not increased at least twice the concurrent negative controls in a dose-dependant manner and the findings were rather reproducible due to similar result in the dose selection test. Further, bacterial effect of the test substance was noted at the dose of 5000 µg/plate in the tester strains in the absence of the metabolic activation system. But, precipitations of the test substance were not noted. Furthermore, the numbers of revertant colonies of the positive controls of all tester strains were remarkably increased compared to their respective negative controls.

The findings concluded that HBM-AV had no mutagenicity under this experimental condition.

Chromosome Aberration (Zheng MS, 2007): Negative result

This study was designed to examine a clastogenic potential of 2-(2-Vinyloxyethoxy)ethyl acrylate in the chromosome aberration test system using Chinese hamster lung cell (CHL/IU).

The result of growth inhibition test showed that the IC50 (Inhibition concentration 50%) was calculated as 9.2 µg/mL and 422.1 µg/mL for the short time treatment without and with metabolic activation system and 8.9 µg/mL for the continuous treatment. On the basis of the growth inhibition test, the highest dose in chromosome aberration test was chosen as 1 0 µg/mL for the short time treatment without metabolic activation system and the continuous treatment, and 420 µg/mL for the short time treatment with metabolic activation system. Subsequently, the highest dose formulations were diluted by common ratio of 2 to produce 2 additional lower doses levels accompanied by a negative and positive control.

Each dosing formulations were treated for 6 and 24 hours for the short time treatment regardless of metabolic activation and continuous treatm~nt without metabolic activation, respectively; and specimens were prepared to examine chromosome aberration.

As the result of chromosome aberration test, the number of structural and numerical chromosome aberration cells of treatment groups was not increased as compared with that of a negative control group regardless of application of metabolic activation system in the short time treatment and continuous treatment.

From that the result showed negative for the short time treatment with metabolic activation, the confirmation test was performed to verify the negative result by 6 hours treatment and 42 hours recovery.

Through the confirmation test, it was also found that the number of structural and numerical chromosome aberration for the short time treatment with metabolic activation system was not increased as compared with that of a negative control. It is confirmed that averages of chromosome aberration cells in negative and positive control group were within the range of the historical control data in ourlaboratory.

In conclusion, 2-(2-Vinyloxyethoxy)ethyl acrylate did not show the chromosome aberrations regardless of application of metabolic activation system in the chromosome aberration test system using Chinese hamster lung cell (CHL/IU), under the conditions of this study.

Chromosome Aberration (Hopker, V, 2006): Positive result

The test item 2-(2-Vinyloxyethoxy)ethyl acrylate, dissolved in deionised water, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in one experiment. The following study design was performed:

With and without S9 mix: Exposure period 4 hrs; Recovery 14 hrs; Preparation interval 18 hrs

In each experimental group two parallel cultures were set up. Per culture at least 100 metaphase plates were scored for structural chromosome aberrations. The highest applied concentration in the pre-test on toxicity (1652 Ng/mL; approx. 8.9 mM) was chosen with regard to the solubility properties of the test item in an appropriate solvent with respect to the current OECD Guideline 473. Dose selection for the cytogenetic experiments was performed considering the toxicity data. In the absence of S9 mix, cytotoxicity was observed at the highest evaluated concentration. In the presence of S9 mix, concentrations showing clear cytotoxicity were not scorable for cytogenetic damage. In the absence and presence of S9 mix, statistically significant and biologically relevant increases in the number of cells carrying structural chromosomal aberrations were observed after treatment with the test item. In addition, in the absence of metabolic activation the number of aberrant cells were increased in a dose-related manner. No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls. Appropriate mutagens were used as positive controls. They induced statistically significant increases (p<0.05) in cells with structural chromsome aberrations.

In conclusion, it can be stated that under the experimental conditions reported, the test item induced structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro. Therefore, 2-(2-Vinyloxyethoxy)ethyl acrylate is considered to be clastogenic in this chromosome aberration test in the absence and presence of S9 mix.

Mouse Lymphoma Assay:

The maximum dose level used was limited by test material induced toxicity. Precipitate of test material was not observed at any of the dose levels in the mutagenicity test. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for hte L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material induced very modest but statisticallly significant dose-related increases in the mutant frequency in the absence and presence of metabolic activation, in both the first and second experiment. However, it should be noted that the responses which exceeded the GEF value were at dose levels where the toxicity was at the very limit of acceptability, were not reproducible between the two experiments and with the exception of the 4 -hour exposure group in the absence of metabolic activation in the first experiment the mutant frequency values observed were within or marginally higher than the acceptable range for vehicle controls. It was therefore considered that responses observed were due to a cytotoxic mechanism rather than a true genotoxic response and were, therefore, considered to have no toxicological significance.

The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

In vivo study results:

Mouse micronucleus (key study; Flanders L):

The study was performed to assess the potential of the test material to produce damage to chromosomes or aneuploidy when administered to mice. The method was designed to comply with the 1997 DECD Guidelines for Testing of Chemicals No.474 "Micronucleus Test", Method 812 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the USA EPA, TSCA and FIFRA guidelines and the Japanese METI/MHLW guidelines for testing of new chemical substances.

A range-finding test was performed to find suitable dose levels of the test material, route of administration and to investigate to see if there was a marked difference in toxic response between the sexes. There was no marked difference in toxicity of the test material between the sexes; therefore the main test was performed using only male mice. The micronucleus test was conducted using the oral route in groups of seven mice (males) at the maximum tolerated dose (MTD) 1500 mg/kg and with 750 and 375 mg/kg as the two lower dose levels. Animals were killed 24 or 48 hours later, the bone marrow extracted, and smear preparations made and stained. Polychromatic (PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei.

Further groups, each of 7 mice, were given a single oral dose of arachis oil or dosed orally with cyclophosphamide (5 mice), to serve as vehicle and positive controls respectively. Vehicle control animals were killed 24 or 48 hours later, and positive control animals were killed after 24 hours.

There were no premature deaths seen in any of the dose groups. Clinical signs were observed in animals dosed with the test material at and above 750 mg/kg in both the 24 and 48-hour groups where applicable, these were as follows: Hunched posture and ptosis.

A modest but statistically significant decrease in the PCE/NCE ratio was observed in the 24-hour 375 mg/kg test material group when compared to the concurrent vehicle control group. However, with no evidence of any statistically significant decreases in the PCE/NCE ratio in any of the higher test material dose groups, the response was considered to be spurious and of no biological relevance. The observation of clinical signs, at and above 750 mg/kg in both the 24 and 48-hour groups where applicable, was taken to indicate that systemic absorption had occurred and exposure to the target tissue had been achieved.

There was no evidence of a significant increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test material when compared to the concurrent vehicle control groups.

The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.

The test material was considered to be non-genotoxic under the conditions of the test.

Mouse micronucleus (supporting study; Kawamura K):

The results indicated that 2-(2-Vinyloxyethoxy) ethyl acrylate did not induce micronuclei under the test conditions which ensured sufficient exposure of animals to the substance. 2-(2-Vinyloxyethoxy) ethyl acrylate was considered to be not clastogenic in vivo.

Justification for classification or non-classification

Two in vitro Chromosome aberration studies have been conducted on this test material. The chromosome aberration report (Hopker, V) has produced genotoxic effects in Chinese hamster lung cells. This is compared to no genotoxic effects being observed in the Zheng MS study.

On close inspection of the Hopker, V study a couple of errors have been noted which may have caused a false positive result.

The following comments have been made regarding this study:

-       The chinese hamster lung V79 cell line was used, this is recognised for providing positive results, and due to this, is not used very often.

-        The second test was not performed due to clastogenicity after 24 hours

-       The dose used for without S9 mix was too high and could have contributed to this result.

The Zheng MS study has provided a negative result, this has used a different cell line as to the report above, however in this study a marginally lower toxicity than normal has been used to produce the negative result (please note this was very marginal). As both reports have minor errors the the negative study has been selected as the key study chromosome aberration study. This has been based on the following reasons:

-       The three reasons stated above

-        Due to the fact there has been no positive result in other in-vitro studies (either the Ames study or the mouse lymphoma study).

In-vivo studies (mouse micronucleus studies) have shown the substance to be non-genotoxic under the conditions of these studies.

Based on a weight of evidence approach, the substance is not classified as a germ cell mutagen based on negative in-vitro studies (Ames, Chromosome Aberration, Mouse Lymphoma Assay) and two negative in-vivo mouse micronucleus studies.