Registration Dossier

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:
2003, July-November, day of application: 2003-08-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
July 21, 1997
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
May 19, 2000
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): Trimethylphenol
- Physical state: solid
- Analytical purity: 99.0 area% (analytical report dated 2003-07-25)
- Impurities (identity and concentrations): no data
- Purity test date: 2003-07-25
- Lot/batch No.: B 3020/03.06.2003
- Expiration date of the lot/batch: no data
- Stability under test conditions: yes, determined analytically
- Storage condition of test material: room temperature, N2 conditions
- Other: yellowish solidified melt

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland GmbH
- Age at study initiation: 5 - 8 weeks
- Weight at study initiation: 29 g (mean)
- Assigned to test groups randomly: yes, computerized randomization plan
- Fasting period before study: no data
- Housing: individually in Makrolon cages, type MI
- Diet: standardized pelleted feed (Maus/Ratte Haltung "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland), ad libitum
- Water ad libitum; from bottles
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): no data; the animals were accommodated in fully air-conditioned rooms in which central air conditioning
- Photoperiod (hrs dark / hrs light): 12/12 (6.00 - 18.00 hours light/18.00 - 6.00 hours dark)

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: olive oil
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water, olive oil was selected as the vehicle, which had been demonstrated to be suitable in the in vivo micronucleus test and for which historical data are available.
- Concentration of test material in vehicle: 10, 20, 30 g/l
- Amount of vehicle (if gavage or dermal): 10 ml/kg bw
- Lot/batch no. (if required): no data
- Purity: no data
Duration of treatment / exposure:
single dose
Frequency of treatment:
single dose
Post exposure period:
one
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 200, 300 mg/kg bw
Basis:
analytical conc.
No. of animals per sex per dose:
5 males/group
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide ((Endoxan®, ASTA MEDICA, Reg. Nr. E 432-1) for clastogenic effects; solvent: water
- Justification for choice of positive control(s): The stability of CPP is well-defined under the selected conditions, since the positive control article is a well-established reference clastogen.
- Route of administration: i.p.
- Doses / concentrations: 20 mg/kg bw

vincristine sulphate (SIGMA - V 8879) for aneugenic effects; solvent water
- Justification for choice of positive control(s): The stability of VCR is well-defined under the selected conditions, since the positive control article is a well-established reference aneugen.
- Route of administration: i.p.
- Doses / concentrations: 0.15 mg/kg bw

Examinations

Tissues and cell types examined:
bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
In a pretest for the determination of the acute intraperitoneal toxicity, deaths were observed down to a dose of 450 mg/kg body weight . 400 mg/kg bw were survived by all animals, but led to clinical signs such as piloerection, squatting posture and the general state of the animals was poor. However, there were no distinct differences in the symptoms between males and females. Thus, only male animals were used for the cytogenetic investigations.
A dose of 400 mg/kg bw was selected as the highest dose in the present cytogenetic study. However, in the main experiment, 3 out of 10 animals died unexpectedly after test substance administration. Therefore the following doses were finally selected for the cytogenetic study: 300 mg/kg, 200 mg/kg and 100 mg/kg body weight.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
At the beginning of the study, the animals were weighed and the substance to be administered or the amount of volume was related to the specific weight of the individual animals on the day of the experiment. All test substance formulations were prepared immediately before administration.
Groups of 5 males were given a single intraperitoneal dose if the test substance, the vehicle or of the positive control substances and were sacrificed at 24 and/or 48 hours after dosing (see freetext below).

The bone marrow was prepared according to the method described by SCHMID W [The micronucleus test for cytogenetic analysis. In : Hollaender, A. (ed), Chemical Mutagens, Principles and Methods for their Detection, Volume 4, Plenum Press, New York (1976 )] and SALAMONE M et al [Mutat Res 74: 347-356 (1980)]
• The two femora of the animals sacrificed by cervical dislocation were prepared by dissection and removing all soft tissues.
• After cutting off the epiphyses, the bone marrow was flushed out of the diaphysis into a centrifuge tube using a cannula filled with fetal calf serum which was at 37°C (about 2 ml/femur).
• The suspension was mixed thoroughly with a pipette, centrifuged at 300 x g for ~ 5 minutes, the supernatant was removed and the precipitate was resuspended in about 50 µl fresh FCS.
• 1 drop of this suspension was dropped onto clean microscopic slides, using a Pasteur pipette. Smears were prepared using slides with ground edges, the preparations were dried in the air and subsequently stained .

DETAILS OF SLIDE PREPARATION:
• The slides were stained in eosin and methylene blue (modified May-Grünwald solution or Wrights solution) for about 5 minutes.
• After having briefly been rinsed in purified water, the preparations were soaked in purified water for about 2 - 3 minutes.
• Subsequently, the slides were stained in Giemsa solution (15 ml Giemsa, 185 ml purified water) for about 15 minutes.
• After having been rinsed twice in purified water and clarified in xylene, the preparations were mounted in Corbit-Balsam.

METHOD OF ANALYSIS:
Microscopic evaluation
In general, 2000 polychromatic erythrocytes (PCEs) from each of the male animals of every test group are evaluated and investigated for micronuclei (MN). The normochromatic erythrocytes (NCEs) which occur are also scored. The following parameters are recorded :
• Number of polychromatic erythrocytes
• Number of polychromatic erythrocytes containing micronuclei
• Number of normochromatic erythrocytes
• Number of normochromatic erythrocytes containing micronuclei
• Ratio of polychromatic to normochromatic erythrocytes
• Number of small micronuclei (d < D/4) and of large micronuclei (d > D/4) (d = diameter of micronucleus, D = cell diameter)
Slides were coded before microscopic analysis. Since the absolute values shown were rounded but the calculations were made using the unedited values, there may be deviations in the given relative values.
Evaluation criteria:
Acceptance criteria:
The mouse micronucleus test is considered valid if the following criteria are met:
• The quality of the slides must allow the identification and evaluation of a sufficient number of analyzable cells, i.e. => 2 000 PCEs and a clear differentiation between PCEs and NCEs.
• The ratio of PCEs/NCEs in the untreated animals (negative control) has to be within the normal range for the animal strain selected.
• The number of cells containing micronuclei in negative control animals has to be within the range of the historical control data both for PCEs and for NCEs.
• The two positive control substances have to induce a significant increase in the number of PCEs containing small and large micronuclei within the range of the historical control data or above.

Assessment criteria:
A finding is considered positive if the following criteria are met:
• Significant and dose-related increase in the number of PCEs containing micronuclei.
• The number of PCEs containing micronuclei has to exceed both the concurrent negative control and the highest value of the historical control range.
A test substance is considered negative if the following criteria are met:
• The number of cells containing micronuclei in the dose groups is not significantly above the negative control and is within the historical control data.
Statistics:
The statistical evaluation of the data was carried out using the program system MUKERN (BASF Aktiengesellschaft).
The asymptotic U test according to MANN-WHITNEY (modified rank test according to WILCOXON) was carried out to clarify the question whether there were significant differences between the control group and dose groups with regard to the micronucleus rate in polychromatic erythrocytes. The relative frequencies of cells containing micronuclei of each animal was used as a criterion for the rank determination for the U test. Significances were identified as follows:
* p >=0.0 5; ** p <=0.0 1

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls valid:
yes
Positive controls valid:
yes

Any other information on results incl. tables

1. MICROSCOPIC EVALUATION

The single intraperitoneal administration of olive oil in a volume of 10 ml/kg body weight led to 1.0 ‰ polychromatic erythrocytes containing micronuclei after the 24-hour sacrifice interval or to 1.0 ‰ after the 48-hour sacrifice interval.

After the single administration of the highest dose of 300 mg/kg body weight, 2.0 ‰ polychromatic erythrocytes containing micronuclei were found after 24 hours and 1 .2 ‰ after 48 hours.

In the two lower dose groups, rates of micronuclei of about 1.6 ‰ (200 mg/kg group) and 1.2 ‰ (100 mg/kg group) were detected after a sacrifice interval of 24 hours in each case.

With 19.5 ‰ the positive control substance cyclophosphamide for clastogenicity led to the expected clear increase in the number of polychromatic erythrocytes containing exclusively small micronuclei.

With 76.7 ‰ the positive control vincristine for spindle poison effects also led to a clearly enhanced number of polychromatic erythrocytes containing micronuclei with the expected amount of large micronuclei, i.e. 18.1 ‰.

The number of normochromatic erythrocytes containing micronuclei did not differ to any appreciable extent in the negative control or in the various dose groups at any of the sacrifice intervals.

Thus, the test substance 2,3,6-trimethylphenol did not lead to any increase in the rate of micronuclei. The number of normochromatic or polychromatic erythrocytes containing small micronuclei (d < D/4) or large micronuclei (d > D/4) did not deviate from the vehicle control value at any of the sacrifice intervals and was within the historical control range.

No inhibition of erythropoiesis induced by the treatment of mice with 2,3,6 -trimethylphenol

was detected.

2 . CLINICAL EXAMINATIONS

The single intraperitoneal administration of the vehicle in a volume of 10 ml/kg body weight was tolerated by all animals without any signs or symptoms.

The administration of the test substance led to evident signs of toxicity.

Neither the single administration of the positive control substance cyclophosphamide in a dose of 20 mg/kg body weight nor that of vincristine in a dose of 0 .15 mg/kg body weight caused any signs of toxicity.

Applicant's summary and conclusion

Executive summary:

REPORT SUMMARY

The substance 2,3,6 -trimethylphenol was tested for chromosomal damage (clastogenicity) and for the ability to induce spindle poison effects (aneugenic activity) in NMRI mice using the micronucleus test method. For this purpose, the test substance, dissolved in olive oil, was administered once intraperitoneally to male animals at dose levels of 100 mg/kg, 200 mg/kg and 300 mg/kg body weight in a volume of 10 ml/kg body weight in each case. As a negative control, male mice were administered merely the vehicle, olive oil, by the same route, which gave frequencies of micronucleated polychromatic erythrocytes within the historical control range.

Both of the positive control chemicals, i.e. cyclophosphamide for clastogenicity and vincristine for spindle poison effects, led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei.

Animals which were administered the vehicle or the positive control substances cyclophosphamide or vincristine did not show any clinical signs of toxicity. The administration of the test substance led to evident signs of toxicity.

The animals were sacrificed and the bone marrow of the two femora was prepared 24 and 48 hours after administration in the highest dose group of 300 mg/kg body weight and in the vehicle controls. In the test groups of 200 mg/kg and 100 mg/kg body weight and in the positive control groups, the 24-hour sacrifice interval was investigated only. After staining of the preparations, 2000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes with and without micronuclei occurring per 2000 polychromatic erythrocytes were also recorded.

According to the results of the present study, the single intraperitoneal administration of 2,3,6 -trimethylphenol did not lead to any increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was always close to the range as that of the concurrent negative control in all dose groups and at all sacrifice intervals and within the range of the historical control data. No inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected.

Thus, under the experimental conditions chosen here, the test substance 2,3,6 -

trimethylphenol does not have any chromosome-damaging (clastogenic) effect, and there were no indications of any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo.