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Toxicological information

Genetic toxicity: in vivo

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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:
05-27 February 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
The study was conducted according to UKEMS Guidelines for Mutagenicity testing and according to GLP principles. The UKEMS Guidelines for Mutagenicity testing comply with OECD guideline No 474 Micronucleus test, Method B12 of the EEC Commission Directive 2000/32/EC, the USA EPA, TSCA and FIFRA guidelines and the Japanese METI/MHLW guidelines for testing of new chemical substances.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: UKEMS Sub-committee on Guidelines for Mutagenicity Testing, Report, Part 1 revised (Basic Mutagenicity Tests: UKEMS recommended procedures, 1990)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Vinylene Carbonate
- Physical state: clear colourless liquid
- Analytical purity: 99.9%
- Purity test date: 7 January 2002
- Lot/batch No.: 011220
- Storage condition of test material: approx. 4 ºC in the dark, under nitrogen

Test animals

Species:
mouse
Strain:
other: Crl:CD-1(ICR)BR
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent
- Age at study initiation: approx. 5-8 weeks
- Weight at study initiation: 25-30g
- Assigned to test groups randomly: yes
- Fasting period before study: no data
- Housing: in groups of up to 7 in solid-floor polypropylene cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: minimum 7 days
- range finding study: males and females used
- main study: only males used

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12


Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: arachis oil BP
- Justification for choice of solvent/vehicle: no data
- Concentration of test material in vehicle: 0, 15, 30 and 60 mg/ml
- Amount of vehicle (if gavage or dermal): 10 ml/kg
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
freshly as a solution at the appropriate concentration in arachis oil

In the range-finding toxicity study, 2 male and 2 femals mice were also dosed via the intraperitoneal route.

Duration of treatment / exposure:
24 or 48 hrs
Frequency of treatment:
once
Post exposure period:
None.
Doses / concentrations
Remarks:
Doses / Concentrations:
150, 300 and 600 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
Range-finding toxicity study: 2
Main study: 7
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: oral gavage
- Doses / concentrations: 5 mg/ml solution in distilled water. Dose is 50 mg/kg.
- No. of animals dosed: 5

Examinations

Tissues and cell types examined:
micronucleated polychromatic erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:


TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):


DETAILS OF SLIDE PREPARATION:
Immediately following termination (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 and stained in May-Grunwald/Giemsa, allowed to air-dry and coverslipped using mounting medium.

METHOD OF ANALYSIS:
Stained bone marrow smears were coded and examinated blind using light microscopy at x1000 magnification. The incidence of micronucleated cells per 2000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal were scored. In addition, the number of normochromatic erythrocytes (NCE-pink stained mature cells) accociated with 1000 erythrocytes were counted; these cells were also scored for incidence of micronuclei.

OTHER:
Evaluation criteria:
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, dose-responsive, 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 correcsponding control group.
If these criteria were not demonstrated, then the test material was considered to be non-genotoxic under the conditions of the test.
A positive control 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:
Where appropriate all data were statistically analysed using appropriate statistical methods as recommended by the UKEMS sub-committee on Guidelines for Mutagenicity Testing Report, Part III. The data was analysed 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:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY: toxicity study
- Dose range: 600, 800, 1000 and 2000 mg/kg
- deaths and clinical signs of toxicity in test animals: In animals dosed with test material via the intraperitoneal route premature deaths occurred at 2000 mg/kg within one hour of dosing.
In animals dosed with the test material via the oral route premature deaths occurred at 2000 mg/kg, and animals were killed in extremis, due to the severity of their clinical signs, at and above 800 mg/kg. Clinical signs were observed at and above 600 mg/kg and included as follows: hunched posture, lethargy, ptosis, decreased respiratory rate, laboured respiration, pilo-erection, ataxia, gasping respiration, hypothermia, distended abdomen and body tremors.
- difference male/female: The test material showed no marked difference in its toxicity to male and female mice, it was therefore considered to be acceptable to use males only for the main study.
- oral/intraperitoneal route: Adequate evidence of test material toxicity was demonstrated voa the oral route of administration, therefore, this was selected for use in the main study.
- dose main study: the maximum tolerated dose of the test material, 600 mg/kg, was selected for use in the main study, with 150 and 300 mg/kg as the lower dose levels.


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): There were no statistically significant increases in the frequency of micronucleated PCEs in any of the test material dose groups when compared to their concurrent vehicle control groups.
- Ratio of PCE/NCE (for Micronucleus assay): there were no statistically significant decreases in the PCE/NCE ratio in the 24 or 48-hour test material groups when compared to their concurrent vehicle control groups. However, the observation of clinical signs was taken to indicate that systemic absorption had occurred.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative
The test material was considered to be non-genotoxic under the conditions of the test.
Executive summary:

In vivo chromosome aberration potential for Vinylene carbonate was studied in mice orally dosed once at 150, 300 or 600 mg/kg bw in accordance with OECD 474. Reliable positive and negative controls were included.

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