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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Type of genotoxicity: DNA damage and/or repair
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 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
GLP compliance:
yes
Type of assay:
unscheduled DNA synthesis

Test material

Constituent 1
Chemical structure
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.: 107450 09T0
- Storage condition of test material: room temperature, protection from light

Test animals

Species:
rat
Strain:
other: Wistar Han rats, Crl:WI (HAN)
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH
- Age at study initiation: 8-10 weeks
- Weight at study initiation: 243.3 g
- Assigned to test groups randomly: yes
- Fasting period before study: yes (at least 6 hours)
- Housing: individual
- Diet : Standardized pelleted feed (Maus/Ratte Haltung "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water: ad libitum; drinking water from bottles
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of vehicle: suitable in the in vivo UDS assay and historical control data are available
- Concentration of test material in vehicle: 1000 and 2000 mg/10 mL corn oil
- Amount of vehicle: 10 mL/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The substance to be administered per kg body weight was emulsified in corn oil. To achieve homogeneity of the test substance in the vehicle, the test substance preparation was shaken thoroughly. All test substance formulations were prepared immediately before administration.
Duration of treatment / exposure:
3 and 14 h
Frequency of treatment:
once by oral gavage
Post exposure period:
cultivation of hepatocytes overnight until fixation.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
1000 mg/kg bw
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
2000 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
6 ( 3 for the resp. sampling times)
Control animals:
yes, concurrent vehicle
Positive control(s):
- substance: 2-acetylaminofluorene
- Route of administration: oral by gavage
- Doses: 50 mg/kg bw

Examinations

Tissues and cell types examined:
primary rat hepatocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
For genotoxicity investigations it is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly. The test doses were selected in accordance with the requirements set forth in the test guidelines and based on the results of preliminary range finding testing (experimental conduct in accordance with GLP, without a GLP status).

DETAILS OF SLIDE PREPARATION:
After an attachment period of at least 2 hours, the attachment medium (WMEC) was drawn off to remove nonadherent cells and was rinsed with WMEI or HBSS. Subsequently, 2 mL labeling solution containing 3H-thymidine was added to the wells. Then the cultures were incubated at 37°C, 5% (v/v) CO2 and ≥ 90% relative humidity for 4 hours. At the end of the labeling period, the wells were rinsed with WMEI or HBSS. Subsequently, about 2 mL unlabeled thymidine solution was added and the cells were incubated for at least 12 hours at 37°C, 5% (v/v) CO2 and ≥ 90% relative humidity.
Cells were fixed with ethanol/acetic acid (ratio 3:1; v/v) for at least 30 minutes and then rinsed 2 - 4 times with distilled water.
The air-dried coverslips were mounted cell-side-up on glass slides using Corbit-Balsam TM.

METHOD OF ANALYSIS:
The slides were immersed in undiluted Kodak nuclear track emulsion NTB at about 37°C for about 5 – 10 seconds, dried at room temperature overnight and subsequently stored in the dark with a desiccant (in the presence of a drying agent) at about -20°C for at least 3 days.
Thereafter, the slides were left at room temperature for at least 3 hours and then they were treated with Kodak D-19 developing solution at about 15°C for 3 - 5 minutes. Immediately afterward, the developing solution was rinsed with water. The rinsed slides were fixed in Kodak fixer for about 5 minutes. Subsequently, the slides were rinsed with running deionised water for about 5 – 10 minutes.
The slides were stained with hematoxylin-eosin and after having dried in the air, they were covered with a second coverslip using Corbit-Balsam TM.
In general, only 3 out of 6 slides prepared per test group were initially developed autoradiographically.
Evaluation criteria:
Acceptance criteria

The in vivo UDS test is considered valid if the following criteria are met:
• Viability (trypan blue vital dye-exclusion method) of at least 70% in liver cells from the vehicle control animals.
• The quality of the slides must allow the evaluation of a sufficient number of analyzable cells; i. e. ≥ 300 cells per test group.
• The mean net nuclear grain count (NNGC) of the negative controls (vehicle controls) has to be below zero and within the range of the historical negative control data.
• The mean net nuclear grain count (NNGC) of the positive control group has to be distinctly increased compared to the concurrent vehicle control group. The values should be clearly above zero and they should be within the range of the historical positive control data or above. In addition, the percentage of cells in repair should be distinctly increased compared with the concurrent vehicle control group.


Assessment criteria

A test substance is considered positive if both following are met:
• The mean net nuclear grain count (NNGC) must exceed zero at one of the dose groups.
• The mean net nuclear grain count (NNGC) clearly exceeds the value of the concurrent vehicle control group at one of the dose groups.

Statistical significance may give further evidence for a positive evaluation. However, both biological relevance and statistical significance should be considered together. A dose-related increase of the percentage of cells in repair (NNGC ≥ 5) with values of ≥ 20%, and a dose-related increase in the mean net nuclear grain count (NNGC) of about zero is considered to be an indication for a marginal response which needs to be confirmed/clarified in a further experiment.

A test substance is considered negative if the following criteria are met:
• In all dose groups, the mean net nuclear grain counts (NNGC) are close to the values of the concurrent vehicle control group and within the range of the historical negative control data.
Statistics:
Due to the clearly negative findings, a statistical evaluation was not carried out.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid

Any other information on results incl. tables

The administration of 1000 mg/kg and 2000 mg/kg body weight p-Cresolmethylether led to clinical signs of toxicity at both sacrifice intervals. Clinical signs of toxicity in test animals were piloerection and squatting posture in both dose groups after 14 h and a poor general state in animals of the high dose group after 14 h. However, no reduced viability of hepatocytes as indication for test substance induced toxicity was observed.

In both parts of the study, the mean net nuclear grain counts per animal of the test substance treated dose groups (-1.97 to -4.09) were close to the respective vehicle control values (-2.31 to -5.17). In addition, the mean values of the net nuclear grain counts of the test substance treated dose groups (-2.48 to -3.32) were close to the respective vehicle control values (-3.68 to -3.83) and within our historical negative control data range (-1.96 to -7.92)

Applicant's summary and conclusion