3,20-bis(ethylenedioxy)pregna-5,7-diene

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 December 2012 to 10 February 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Performed according to OECD test guideline and GLP compliant.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

There were humidity variations. However, based on laboratory historical data these deviations are considered not to affect the study integrity.

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

3,20-bis(ethylenedioxy)pregna-5,7-diene

EC Number:

243-175-0

EC Name:

3,20-bis(ethylenedioxy)pregna-5,7-diene

Cas Number:

19592-55-3

Molecular formula:

C25H36O4

IUPAC Name:

(1S,3aR,9aR,9bS,11aS)-9a,11a-dimethyl-1-(2-methyl-1,3-dioxolan-2-yl)-1,2,3,3a,6,8,9,9a,9b,10,11,11a-dodecahydrospiro[cyclopenta[a]phenanthrene-7,2'-[1,3]dioxolane]

Test material form:

solid: crystalline

Details on test material:

Slightly yellow powder.

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

NMRI BR mice (SPF) were used as the test system. These mice are recommended by international guidelines (e.g. OECD, EC). Females were nulliparous and non-pregnant. The animals were provided by Charles River, Sulzfeld, Germany.

Young adult animals were selected (6-7 weeks old at the start of treatment). The total number of animals used in the dose range finding study was 10 and in the main study 60. In the micronucleus main study 5 male and 5 female mice were treated per sampling time in each treatment group.

The body weights of the mice at the start of the treatment were within 20% of the sex mean. The mean body weights were for males 35.1 ± 1.8 g and for females 26.6 ± 1.0 g and the range was for males 32 – 38 g and for females 24 - 28 g. The animals were allocated at random to the treatment groups.

The acclimatisation period was at least 6 days before the start of treatment under laboratory conditions.

Conditions
A controlled environment was maintained in the room with optimal conditions of approximately 15 air changes per hour, a temperature of 21.0 ± 3.0°C (actual range: 20.6 - 21.4°C), a relative humidity of 40 - 70% (actual range: 32 - 52%) and a 12 hour light/12 hour dark cycle. Deviations in the minimum level of humidity are explained in the deviation section above.

Accommodation
The animals were group housed (maximum 5 animals per sex per cage) in labelled Macrolon cages (type MIII height: 15 cm) containing sterilised sawdust as bedding material (Litalabo; S.P.P.S., Argenteuil, France). A shelter (disposable paper corner home, MCORN 404, Datesand Ltd, USA) and paper bedding (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom) was provided as cage-enrichment.

Diet
The animals had free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).

Water
The animals had free access to tap-water.

Diet, water, bedding and cage enrichment evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Corn oil (Sigma, Zwijndrecht, The Netherlands).

Details on exposure:

Proketal was suspended in corn oil (Sigma, Zwijndrecht, The Netherlands). The specific gravity of corn oil is 0.9 g/ml. Proketal concentrations were treated with ultra-sonic waves to obtain a homogeneous suspension.

Duration of treatment / exposure:

Animals were dosed twice with approximately one hour interval between treatments. Based on the results of the pilot study, mice of the main study received an oral intubation of a maximum tolerated (high), an intermediate and a low dose of Proketal. To obtain the recommended highest dose level according to the guidelines, mice received two consecutive doses with one hour interval (100 mg/ml; 10 ml/kg).

Frequency of treatment:

Twice (see above- duration of treatment/exposure)

Post exposure period:

The observation period after dosing was one to 3 days. During this period mortality and physical condition were recorded at least once a day.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 500, 1000, 2000 mg/kg/bw
Basis:
nominal conc.
oral gavage

No. of animals per sex per dose:

3/sex/group

Control animals:

yes, concurrent vehicle

Positive control(s):

The positive control used in the micronucleus test was cyclophosphamide (CAS no. 50-18-0; Baxter B.V., Utrecht, The Netherlands) dissolved in physiological saline (B. Braun, Melsungen AG, Germany) dosed as a single oral intubation of 40 mg/kg body weight.

The route and the volume administered of the positive control were the same as those of the test substance.

Examinations

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

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 remaining serum. A drop of the cell suspension was placed on the end of a clean slide, which was previously immersed in a 1:1 mixture of 96% (v/v) ethanol (Merck, Darmstadt, Germany)/ether (Merck) and cleaned with a tissue. The slides were marked with the study identification number and the animal number. The drop was spread by moving a clean slide with round-whetted 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 (Merck) and air-dried overnight. Two slides were prepared per animal.

Evaluation criteria:

To prevent bias, all slides were randomly coded before examination. An adhesive label with the 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 at least 2000 polychromatic erythrocytes (with a maximum deviation of 5%). The ratio of polychromatic to normochromatic erythrocytes was determined by counting and differentiating at least the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were calculated.

Statistics:

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, one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time) in the combined data for both sexes or in the data for male or female groups separately and the number of micronucleated polychromatic erythrocytes in the animals are above the historical control data range.

A test substance is considered negative in the micronucleus test if:
- None of the tested concentrations or sampling times showed a statistically significant (Wilcoxon Rank Sum Test, one-sided, p < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes either in the combined data for both sexes or in the data for male or female groups separately and the number of micronucleated polychromatic erythrocytes in the animals are within the historical control data range.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

All animals treated with Proketal and the animals of the negative and positive control groups showed no treatment related clinical signs of toxicity or mortality.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
It is concluded that this test is valid and that Proketal is not clastogenic or aneugenic in the bone marrow micronucleus test when sampled at 24 and 48 hours post dosing of male and female mice up to a dose of 2000 mg/kg (the maximum recommended dose in accordance with current regulatory guidelines) under the experimental conditions described in this report.