4-methylanisole

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: According to GLP and current testing guidelines.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

4-methylanisole

EC Number:

203-253-7

EC Name:

4-methylanisole

Cas Number:

104-93-8

Molecular formula:

C8H10O

IUPAC Name:

1-methoxy-4-methylbenzene

Details on test material:

- Name of test material (as cited in study report): p-Cresolmethylether
- Physical state: liquid, colorless, clear
- Analytical purity: 98.6%
- Batch No.: 10745009T0
- Storage condition of test material: room temperature, protection from light
- Expiration Date: 2010-07-31

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories GmbH, 33178 Borchen, Germany
- Age at study initiation: 8 weeks
- Weight at study initiation: mean value 38.8 g (SD +/- 3.4 g)
- Assigned to test groups randomly: yes
- Housing: single, in Makrolon Type II/III, with wire mesh top (EHRET GmbH, 79302 Emmendingen, Germany)
- Diet: pelleted standard diet, ad libitum (Harlan Laboratories GmbH, 33178 Borchen, Germany)
- Water: ad libitum; tap water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 45 - 65
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: corn oil
- Justification for choice of vehicle: The vehicle was chosen due to its relative non-toxicity for the animals.
- Concentration of test material in vehicle: 500, 1000, and 2000 mg/kg b.w.
- Amount of vehicle: 10 mL/kg bw.

Details on exposure:

The animals received the test item, the vehicle or the positive control substance once orally

Duration of treatment / exposure:

24 and 48 h

Frequency of treatment:

single daily administration

Post exposure period:

Not applicable

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
24 h preparation interval: 500, 1000 and 2000 mg/kg b.w.
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
48 h preparation interval: 2000 mg/kg b.w.
Basis:
nominal conc.

No. of animals per sex per dose:

7 males in each dosing group.
5 males in the vehicle and positive control groups.

Control animals:

yes, concurrent vehicle

Positive control(s):

40 mg/kg bw cyclophosphamide

Examinations

Tissues and cell types examined:

Polychromatic/Normochromatic erythrocytes (PCE) in the bone marrow

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION:
The animals were sacrificed using CO2 followed by bleeding. The femora were removed, the epiphyses were cut off and the marrow was flushed out with foetal calf serum using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the re-suspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald (Merck, 64293 Darmstadt, Germany)/Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (Kindler, 79110 Freiburg, Germany). At least one slide was made from each bone marrow sample.

Analysis of Cells
Per animal 2000 polychromatic erythrocytes (PCE) were analysed for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides.

Evaluation criteria:

The study was considered valid as the following criteria are met:
- at least 5 animals per group can be evaluated.
- PCE to erythrocyte ratio should not be less than 20 % of the negative control.
- the positive control shows a statistically significant and biological relevant increase of micronucleated PCEs compared to the vehicle control.

Evaluation of Results
A test item is classified as mutagenic if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group. Statistical methods are used as an aid in evaluating the results, if necessary. However, the primary point of consideration is the biological relevance of the results. A test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.

Statistics:

Nonparametric Mann-Whitney test

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Any other information on results incl. tables

Since no gender specific differences concerning signs of toxicity in the pre-experiment were observed, the main study was performed using males only.

Clinical signs of toxicity, i.e. ruffled fur, were observed in all test substance dose groups and reduction of spontaneous activity was observed in the high dose animals.

The mean number of polychromatic erythrocytes (PCEs) was not decreased after treatment with the test item as compared to the mean value of PCEs of the vehicle control indicating that p-Cresolmethylether did not have any cytotoxic properties in the bone marrow.

In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item.

Test group	Dose (mg/kg bw)	Sampling time (h)	PCEs with micronuclei (%)	PCE per 2000 erythocytes
Vehicle	0	24	0.150	1181
Test item	500	24	0.100	1196
Test item	1000	24	0.079	1207
Test item	2000	24	0.114	1141
Positive ctrl.	40	24	2.840	1155
Vehicle	0	48	0.070	1232
Test item	2000	48	0.064	1030

Applicant's summary and conclusion

Conclusions:

It can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, p-Cresolmethylether is considered to be non-mutagenic in this micronucleus assay.

Executive summary:

The ability of p-Cresolmethylether to induce micronuclei in polychromatic erythrocytes (PCE) was investigated in the bone marrow of mice. The test item was fomulated in corn oil (vehicle control) and administered by gavage at :24 h preparation interval: 500, 1000, and 2000 mg/kg b.w and 48 h preparation interval: 2000 mg/kg b.w. Postive controls recieved 40 mg/kg bw cyclophosphamide orally. Seven males per test group (except the vehicle and positive control groups with 5 males each) were evaluated for the occurrence of micronuclei. Per animal 2000 polychromatic erythrocytes (PCEs) were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes. The highest dose (2000 mg/kg; maximum guideline-recommended dose) was estimated by a pre-experiment to be suitable. Since no gender specific differences concerning signs of toxicity in the pre-experiment were observed, the main study was performed using males only. After treatment with the test item the number of PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control thus indicating that pCresolmethylether did not exert any cytotoxic effects in the bone marrow. In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used. Under the experimental conditions reported, p-Cresolmethylether is considered to be non-mutagenic in this micronucleus assay.