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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 July 2002 to 19 July 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: USA EPA, TSCA and FIFRA guidelines
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese METl/MHLW guidelines
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: in vivo mouse micronucleus

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
439-910-1
EC Name:
-
Cas Number:
93705-66-9
Molecular formula:
C23H28O4
IUPAC Name:
2,2'-(methylenebis((2,6-dimethyl-4,1-phenylene)oxymethylene))bis oxirane
Test material form:
solid: particulate/powder
Details on test material:
- Appearance: cream coloured powder
- Storage: room temperature, in the dark until 08 July 2002 and then approximately 4°C, in the dark

Test animals

Species:
mouse
Strain:
CD-1
Remarks:
Crl:CD-1™(ICR)BR
Details on species / strain selection:
Using existing toxicity data there was no marked difference in test material toxicity between the sexes, therefore the study was performed using only male mice.
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: five to eight weeks old
- Weight at study initiation: 25 to 30 g
- Assigned to test groups randomly: the animals were selected at random and given a number unique within the study by ear punching and a number written on a colour coded cage card
- Housing: The animals were housed in groups of up to seven in solid-floor polypropylene cages with wood-flake bedding
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: a minimum of seven days

ENVIRONMENTAL CONDITIONS
- Temperature: 19 - 25°C
- Humidity: 30 - 70%
- Air changes: approximately fifteen changes per hour
- Photoperiod: the lighting was controlled by a time switch to give twelve hours light and twelve hours darkness

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: arachis oil
Duration of treatment / exposure:
A single dose administration
Frequency of treatment:
Once
Post exposure period:
One group of mice from each dose level was killed by cervical dislocation 24 hours following treatment and a second group dosed with 2000 mg/kg was killed after 48 hours.
Doses / concentrationsopen allclose all
Dose / conc.:
2 000 mg/kg bw (total dose)
Dose / conc.:
1 000 mg/kg bw (total dose)
Dose / conc.:
500 mg/kg bw (total dose)
No. of animals per sex per dose:
Seven maless per dose. An additional seven males were treated at 2000 mg/kg for termination after 48 hours.
Control animals:
yes, concurrent vehicle
Positive control(s):
- Cyclophosphamide
- 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.

Examinations

Tissues and cell types examined:
Slides were prepared from the bone marrow of the femora and stained. The numbers of micronucleated polychromatic erythrocytes and normochromatic erythrocytes were recorded.
Details of tissue and slide preparation:
RANGE-FINDING STUDY
- A range-finding toxicity study was performed to determine a suitable dose level and route of administration for the micronucleus study. The dose level selected should ideally be the maximum tolerated dose level or that which produces some evidence of toxicity up to a maximum recommended dose of 2000 mg/kg. Due to limitations of the test material formulation it was not possible to achieve 2000 mg/kg via the intraperitoneal route. Using existing toxicology data it was considered to be acceptable to only use male animals in the study.
- Two males were dosed at 2000 mg/kg (oral) and two males were dosed at 1000 mg/kg (i.p.). All animals were dosed once only at the appropriate dose level by gavage using a metal cannula or with a hypodermic needle attached to a graduated syringe. The volume administered to each animal was calculated according to its bodyweight at the time of dosing.
- Animals were observed one hour after dosing and subsequently once daily for two days. Any deaths and evidence of overt toxicity were recorded at each observation. No necropsies were performed.

MICRONUCLEUS STUDY
- Groups, each of seven mice, were dosed once only via the oral route with the test material at 500, 1000 or 2000 mg/kg. One group of mice from each dose level was killed by cervical dislocation 24 hours following treatment and a second group dosed with 2000 mg/kg was killed after 48 hours. In addition, three further groups of mice were included in the study; two groups (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, a positive control material known to produce micronuclei under the conditions of the test. The vehicle controls were killed 24 or 48 hours following dosing and positive control group animals were killed 24 hours following dosing.
- 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.

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 and stained in May-Grünwald/Giemsa, allowed to air-dry and cover-slipped using mounting medium.

SLIDE EVALUATION
- 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 were 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.
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, 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 corresponding 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 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.
- 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
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING TOXICITY STUDY
- In animals dosed with test material via the intraperitoneal route there were no premature deaths but clinical signs were observed at 1000 mg/kg as follows: hunched posture and lethargy.
- In animals dosed with the test material via the oral route there were no premature deaths, however, clinical signs were observed at 2000 mg/kg as follows: hunched posture.
- There was little difference between the dose routes in the toxic response and the test material suspension was more suited to dosing via the oral route. Results from the 28-day oral toxicity study indicated clinical effects were induced following oral administration. It was, therefore, considered that adequate exposure would be achieved via the oral route. Therefore, the oral route was selected for use in the main study. The maximum recommended dose (MRD) of the test material, 2000 mg/kg, was selected for use in the main study, with 1000 and 500 mg/kg as the lower dose levels.

MICRONUCLEUS 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 2000 mg/kg in both the 24 and 48-hour groups where applicable, these were as follows: hunched posture.

Evaluation of Bone Marrow Slides
- 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.
- 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.
- 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:
Under the conditions of this study the test material was considered to be non-genotoxic.
Executive summary:

The genetic toxicity of the test material was investigated in accordance with the standardised guidelines OECD 474, EU Method B.12., the USA EPA, TSCA and FIFRA guidelines and the Japanese METl/MHLW guidelines for testing of new chemical substances. The method was designed to comply with the UKEMS Sub-committee on Guidelines for Mutagenicity Testing, Report, Part 1 revised (Basic Mutagenicity Tests: UKEMS recommended procedures, 1990). The testing was conducted under GLP conditions.

A range-finding study was performed to find suitable dose levels of the test material and route of administration. Using existing toxicity data there was no marked difference in test material toxicity between the sexes, therefore the study was performed using only male mice. The micronucleus study was conducted using the oral route in groups of seven mice at the maximum recommended dose (2000 mg/kg) with 1000 and 500 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 of mice were given a single oral dose of arachis oil (7 mice) 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.

Under the conditions of the study no statistically significant decreases in the PCE/NCE ratio were observed in the 24 or 48-hour test material dose groups when compared to their concurrent control groups. 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 material produced a marked increase in the frequency of micronucleated polychromatic erythrocytes.

The test material was therefore considered to be non-genotoxic.