2-(2-oxo-5-(1,1,3,3-tetramethylbutyl)-2,3-dihydro-1-benzofuran-3-yl)-4-(1,1,3,3-tetramethylbutyl)phenyl acetate

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:

07.03. - 25.03.2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline study, GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted 21st July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Remarks:

NOTOX B.V., Hambakenwetering 7, 5231 DD ‘s-Hertogenbosch, The Netherlands

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

431-770-1

EC Name:

-

Cas Number:

216698-07-6

Molecular formula:

C32 H44 O4

IUPAC Name:

2-[2-oxo-5-(2,4,4-trimethylpentan-2-yl)-2,3-dihydro-1-benzofuran-3-yl]-4-(2,4,4-trimethylpentan-2-yl)phenyl acetate

Details on test material:

- Description: White solid
- Purity: 98.7%
- Storage condition of test material: At room temperature in the dark

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 6 weeks
- Weight at study initiation: between 30 and approx. 33 g
- Housing: Five male animals per group were housed in labeled polycarbonate cages containing purified sawdust as bedding material (SAWI, Jelu Werk, Rosenberg, Germany).
- Diet: standard pelleted laboratory animal diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: At least 5 days before start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 °C
- Humidity (%): 30-70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

Vehicle used: Corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was suspended in corn oil. Test substance concentrations were blended and treated by ultra sonication to obtain a homogeneous suspension. Test substance concentrations were dosed within 4 hours after preparation.

Duration of treatment / exposure:

A limit study with a repeated dose (with a 24 h interval) of 1000 mg/kg bw with male mice only was performed and sampled (after the second dosing)

Frequency of treatment:

Since, the test substance was difficult to suspend in corn oil, the highest dose that could be reached was 1000 mg/kg body weight. Therefore, the mice received a repeated intraperitoneal injection (with a 24 h interval) of 1000 mg/kg body weight to reach the limit dose of 2000 mg/kg body weight.
The route of administration was chosen to maximize the chance of the test substance reaching the target tissue. The dosing volume was 10 mL/kg bw.

Doses / concentrations

Remarks:

Doses / Concentrations:
approx. 2000 mg/kg bw (2x 1000 mg/kg bw)
Basis:
nominal conc.

No. of animals per sex per dose:

Two groups of each 5 males.

Control animals:

yes, concurrent vehicle

Positive control(s):

The positive control used was cyclophosphamide dissolved in 0.9% (w/v) NaCl in Milli-RO water dosed at a single intraperitoneal injection of 50 mg salt/kg bw.

Examinations

Tissues and cell types examined:

Bone marrow smears from femur.

Details of tissue and slide preparation:

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
The animals dosed with corn oil and the test substance were sacrificed by cervical dislocation 24 or 48 h after the second dosing. The animals treated with cyclophosphamide were sacrificed by cervical dislocation 48 hr after dosing. Both femurs were removed and freed of blood and muscles. Both ends of the bone were shortened until a small opening to the marrow canal became visible. The bone was flushed with approximately 2 ml of foetal calf serum. The cell suspension was collected and centrifuged at 1000 rpm (approximately 100 g) for 5 min. The supernatant was removed with a Pasteur pipette. A drop of serum was left on the pellet. The cells in the sediment were carefully mixed with the serum by aspiration with the remaining serum. A drop of the cell suspension was placed on the end of a slide. The drop was spread by moving a clean slide with roundwhetted sides at an angle of approximately 45° over the slide with the drop of bone marrow suspension. The preparations were air-dried, fixed for 5 min in 100% methanol and air-dried overnight. Two slides were prepared per animal.

DETAILS OF SLIDE PREPARATION:
The slides were automatically stained using the "Wright-stain-procedure" in an "Ames" HEMA-tek slide stainer (Miles, Bayer Nederland B.V.). The dry slides were dipped in xylene before they were embedded in MicroMount and mounted with a coverslip.

METHOD OF ANALYSIS:
All slides were randomly coded before examination. An adhesive label with NOTOX study identification number and code was stuck over the marked slide. At first the slides were screened at a magnification of 100 X for regions of suitable technical quality, i.e. where the cells were well spread, undamaged and well stained. Slides were scored at a magnification of 1000 x. The number of micronucleated polychromatic erythrocytes was counted in 2000 polychromatic erythrocytes. The ratio polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were calculated.

Evaluation criteria:

A test substance is considered positive in the micronucleus test if:
It induced a biologically as well as a statistically significant (Wilcoxon Rank Sum Test; two-sided test at P < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time).
A test substance is considered negative in the micronucleus test if:
None of the tested concentrations or sampling times showed a statistically significant (P < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes.

The incidence of micronucleated polychromatic erythrocytes in the control animals should reasonably be within the laboratory historical solvent control data range; (mean + three times the standard deviation: 0.69%o ± 0.80 %o indicated are means for n=201).

Statistics:

The Wilcoxon rank-sum test was used to assess significant differences between the numbers of micronuclei in the treatment and control groups, in which P<0.05 was used as the lowest level of significance.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE FINDING STUDY
A dose range finding study performed with the test article (NOTOX Project 264397) showed no toxic effect in male or female mice when receiving a repeated (with a 24 h interval) intraperitoneal injection with 1000 mg/kg body weight. Therefore, a limit study with a repeated dose (with a 24 h interval) of 1000 mg/kg body weight with male mice only was performed.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay):
The mean numbers of micronucleated polychromatic erythrocytes scored in the test substance treated groups were compared with the corresponding control groups. No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of test substance treated animals. The incidence of micronucleated polychromatic erythrocytes in the slides of the animals treated with test substance were within the laboratory historical solvent control data range. Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. Hence, the acceptability criteria of the test were met.

- Ratio of PCE/NCE (for Micronucleus assay):
The animals of the groups which were treated with the test substance showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, which reflects a lack of toxic effects of this compound on the erythropoiesis. The group that was treated with cyclophosphamide showed a decrease in the ratio of polychromatic to normochromatic erythrocytes.

- Statistical evaluation: No statistically significant differences in the frequency of erythrocytes containing micronuclei between the solvent control and the dose group (2000 mg/kg bw) was observed.

Any other information on results incl. tables

MEAN NUMBER OF MICRONUCLEATED POLYCHROMATIC ERYTHROCYTES PER 2000 POLYCHROMATIC ERYTHROCYTES AND RATIO OF POLYCHROMATIC/NORMOCHROMATIC ERYTHROCYTES

Group	Treatment	Dose (mg/kg bw)	sampling time	Number of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes (mean ± S.D.)^1	Ratio polychromatic/ normochromatic (mean ± S.D.)
A	corn oil^2	-	24	1.2 ± 0.8	1.01 ± 0.05
B	corn oil^2	-	48	1.4 ± 1.1	1.02 ± 0.06
C	test article^2	1000	24	0.6 ± 0.5	1.00 ± 0.05
D	test article^2	1000	48	1.4 ± 1.7	1.04 ± 0.01
E	CP	50	48	19.6 ± 5.4	0.53 ± 0.21

CP = Cyclophosphamide

¹ Five animals per treatment group

² Dosed twice with a 24 h interval

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
It is concluded that the test article is not mutagenic in the micronucleus test under the experimental conditions described in this report.

Executive summary:

In order to evaluate the test article’s genotoxic effect on erythrocytes in bone marrow, A GLP-compliant Micronucleus Test in mice following OECD guideline 474 was performed. Two groups each comprising 5 males, received a repeated intraperitoneal injection of 1000 mg of the test article per kg body weight with a 24 hours interval. Corresponding vehicle treated groups served as negative controls, a group treated with a single intraperitoneal injection of cyclophosphamide (CP) at 50 mg/kg body weight served as positive control.

Bone marrow was sampled 24 or 48 hours after the second dosing. Bone marrow from the positive control group was harvested at 48 hours after dosing only. Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of animals treated with the test substance compared to the vehicle controls. The incidences of micronucleated polychromatic erythrocytes in the slides of the animals treated with the test substance were within the laboratory historical solvent control data range. The groups that were treated with the test article showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, which reflects a lack of toxic effects of this compound on the erythropoiesis. The group that was treated with cyclophosphamide showed a decrease in the ratio of polychromatic to normochromatic erythrocytes.