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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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well-documented experiment according to a robust test protocol. Only 1 dose tested, however this MTD of 2500 is higher than today's limit dose of 2000 mg/kg and can be considered a limit test; only 1000 instead of 2000 PCE were analysed.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report date:
1991

Materials and methods

Principles of method if other than guideline:
As described below.
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2-hydroxymethyl-9-methyl-6-(1-methylethyl)-1,4-dioxaspiro[4.5]decane
EC Number:
408-200-3
EC Name:
2-hydroxymethyl-9-methyl-6-(1-methylethyl)-1,4-dioxaspiro[4.5]decane
Cas Number:
63187-91-7
Molecular formula:
C13H24O3
IUPAC Name:
[9-methyl-6-(propan-2-yl)-1,4-dioxaspiro[4.5]decan-2-yl]methanol
Test material form:
liquid: viscous

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: BRL Tierfarm Füllinsdorf CH- 4414 Füllinsdorf/Basel, Switzerland
- Age at study initiation: min. 10 weeks
- Weight at study initiation: approx. 30g
- Fasting period before study: yes, approx. 18h
- Housing: Single, in Makrolon Type 1 cages with wire mesh top (EHRET GmbH, D-7830 Emmendingen) and granulated soft wood bedding (ALTROMIN, D-4937 Lage/Lippe)
- Diet: pelleted standard diet (ALTROMIN 1324, D-4937 Lage/Lippe)
- Water: ad libitum
- Acclimation period: min. 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 + 3°C
- Humidity (%): 30-70%
- Air changes (per hr): not reported
- Photoperiod: artificial light 6.00 a.m. - 6.00 p.m.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: polyethylene glycol (PEG 400)
- Justification for choice of solvent/vehicle: non-toxic to the animals
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw
- Supplier: Merck, D-1600 Darmstadt
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulation in PEG 400.
Duration of treatment / exposure:
Single exposure
Frequency of treatment:
Once
Post exposure period:
Sampling of the bone marrow was done 24, 48 and 72 hours after treatment.
Doses / concentrations
Remarks:
Doses / Concentrations:
2500 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5 males and 5 females per test group
Control animals:
yes, concurrent vehicle
Positive control(s):
- Positive control substance: Cyclophosphamide`
- Supplier: SERVA, D-6900 Heidelberg
- Vehicle: physiological saline
- Route of administration: oral gavage
- Doses / concentrations: 30 mg/k gbw

Examinations

Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
A preliminary study on acute toxicity was performed with the same strain and under identical conditions as in the mutagenicity study. In a series of pre-experiments 4 animals (2 males, 2 females) per dose received orally a single dose of the test substance formulated in PEG 400. The volume administered was 10 ml/kg b.w.. The following dose levels of the test article were investigated: 5000, 4000, 3000, 2500 and 2000 mg/kg bw. Observed effects included reduction of spontaneous activity, eyelid closure, apathy, abdominal position and death. No deaths or abdominal position were observed in the 2000 or 2500 mg/kg bw dose group. On the basis of these results 2500 mg/kg b.w. of the test article was estimated to be the maximum tolerated dose.

TREATMENT:
Approximately 18 hours before treatment. with the test article the animals received no food but water ad libitum. At the beginning of the treatment the animals were weighed and the individual volume to be administered was adjusted to the animal's body weight. The animals received the test article once. Twelve
animals, six males and six females, were treated per dose group. Sampling of the bone marrow was done 24, 48 and 72 hours after treatment.

PREPARATION OF THE ANIMALS:
The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a 5 ml syringe. The cell suspension was centrifuged at 1,500 rpm for 10 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread an a slide. The smear was air-dried and then stained with May-Grünwald (MERCK, D-6100 Darmstadt)/Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (KINDLER, D-7800 Freiburg). At least one slide was made from each bone marrow sample.

ANALYSIS OF CELLS:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. 1000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in normochromatic erythrocytes per 1000 the PCEs. The analysis was performed with coded slides. Five animals per sex and group were evaluated as described. The remaining animal of each test group was evaluated in case an animal had died in its test group spontaneously or due to gavage error.

Evaluation criteria:
A test article is classified as mutagenic if it induces a statistically significant increase in the number of micronucleated polychromatic erythrocytes at for at least one of the test points.

A test article producing no statistically significant increase in the number of micronucleated polychromatic erythrocytes at any of the test points is considered non-mutagenic in this system.

This can be confirmed by means of the nonparametric Mann-Whitney test.

However, both biological and statistical significance should be considered together.
Statistics:
Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric mann-Whitney test.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In comparison to the corresponding negative controls there was no enhancement in the frequency of the detected micronuclei at preparation intervals 24 h and 48 h after application of the test article. Biometric analysis of the results at preparation interval 72 h demonstrated a statistically significant difference between negative control and test article group. However, the mean value of 0.15 % cells with micronuclei is not different from the micronucleus frequency after treatment with the test article at preparation interval 24 h and the range of 0 - 2 micronuclei per 1000 cells does not exceed the range of the corresponding negative control. Therefore, the slightly enhanced (as compared to the corresponding negative control) micronucleus frequency after treatment with the test article at preparation interval 72 h is not considered to be of biological relevance.

Any other information on results incl. tables

 

 

24 h

48 h

72h

Group

Dose

(mg/kg bw)

PCE’s with micronuclei

Range

PCE/NCE

PCE’s with micronuclei

Range

PCE/NCE

PCE’s with micronuclei

Range

PCE/NCE

Vehicle

0

0.09%

0-3

1000/667

0.09%

0-3

1000/840

0.07%

0-2

1000/722

Test art.

2500

0.15%

0-3

1000/845

0.09%

0-2

1000/800

0.15%

0-2

1000/575

CPA

30

2.03%

9-34

1000/743

 

 

 

 

 

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
During the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.
Executive summary:

The study investigates the potential of the test substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.

 

The test article was formulated in polyethylene glycol 400 (PEG 400). This vehicle was also used as the negative control. The volume administered orally was 10 mL/kg bw. At 24h, 48h and 72h after a single application of the test article the bone marrow cells were collected for micronuclei analysis. Ten animals (5 males and 5 females) per test group were evaluated for the occurrence of micronuclei. 1000 Polychromatic erythrocytes (PCE) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 1000 PCE. The investigated dose level of the test article was 2500 mg/kg bw.

 

In a series of pre-experiments this dose level was estimated to be the maximum tolerated dose. The animals expressed toxic reactions. After treatment with the test article the ratio between PCEs and NCEs was not substantially affected as compared to the corresponding negative controls, thus indicating no cytotoxic effects. In comparison to the corresponding negative controls, there was no enhancement of biological relevance in the frequency of the detected micronuclei at any preparation interval after application of the test article.

 

An appropriate mutagen was used as positive control, which showed a distinct increase of induced micronucleus frequency.

 

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, the test substance is considered to be non-mutagenic in this micronucleus assay.

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