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

Genetic toxicity: in vivo

Administrative data

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.

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

451-690-9

EC Name:

-

Cas Number:

86273-46-3

Molecular formula:

C9H14O4

IUPAC Name:

2-[2-(ethenyloxy)ethoxy]ethyl prop-2-enoate

Constituent 2

Reference substance name:

2-Propenoic acid 2-(2-Vinyloxyethoxy)ethyl ester

IUPAC Name:

2-Propenoic acid 2-(2-Vinyloxyethoxy)ethyl ester

Test material form:

other: liquid

Details on test material:

Sponsor's identification: VEEA
Description: Clear colourless liquid
Chemical name: 2-(2-Vinyloxyethoxy)ethyl acrylate
Purity: 99.5%
Batch number: 7D23-3323
Date received: 12 December 2008
Storage conditions: Approximately 4°C in the dark

The integrity of supplied data relating to the identity, purity and stability of the test material is the responsibility of the Sponsor.

Test animals

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

Administration / exposure

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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 500 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

750 mg/kg bw/day (nominal)

Doses / concentrations 3

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.

Examinations

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.

Results and discussion

Test results

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.

Applicant's summary and conclusion

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.