Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

in vitro studies
Gene mutation in bacteria
(1) S. typhimurium TA 1535, TA 1537, TA 98, TA100, E. coli WP2 uvr A, with and without metabolic activation: negative (GLP, OECD 471, Miyagawa M 1999)
(2) S. typhimurium TA 1535, TA 1537, TA 98, TA100, with and without metabolic activation: negative (non-GLP, OECD 471 and 472, BASF 1989 Report 40M0513/874186)

Gene mutation in mammalian cells
In Vitro Mammalian Cell Gene Mutation Test, mouse lymphoma L5178Y cells, with and without metabolic activation: Mouse lymphoma assay: negative (GLP, OECD 476, BioReliance 2008)

Cytogenicity in mammalian cells
Chromosome aberration assay in Chinese hamster CHL/IU cells, with and without metabolic activation: positive (GLP, OECD 473, Ohata 1999)



Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
no data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented study report which meets basic scientific principles.
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Target gene:
For Salmonella typhimurium strains, the amino acid histidine locus is the target gene.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
rat liver S-9, induced with Aroclor 1254
Test concentrations with justification for top dose:
20 - 5000 µg/plate; see below
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: no data
Negative controls:
yes
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene, N-methyl-N'-nitro-N-nitroso-guanidine, 4-nitro-o-phenylendiamine, 9-aminoacridine; depending on strain and activation condition
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation) and preincubation;
Evaluation criteria:
In general, a substance to be characterized as positive in the Ames test has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results
Statistics:
no data
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
>= 2500 µg/plate (SPT); >= 1500 µg/plate (PIT)
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

No increase in the number of revertants was observed with any tester strain under any test condition.

Bacteriotoxicity (indicated by reduced bacterial growth) was observed in the SPT at doses >= 2500 µg/plate and in the PIT at 1500 µg/plate.

The test substance was completely soluble in the vehicle chosen; no precipitation was noted at any concentration tested.

Executive summary:

REPORT SUMMARY

The substance 2,3,6-Trimethylphenol was tested for mutagenicity in the Ames test.

Strains: TA 1535, TA 100, TA 1537, TA 98

Dose range: 20 µg - 5000 µg/plate (SPT; TA 100, TA 98)

4 µg - 2500 µg/plate (SPT; TA 1535, TA 1537)

4 µg - 1500 µg/plate (PIT; all tester strains )

Test conditions: Standard plate test ( SPT) and preincubation test ( PIT) both with and without metabolic activation (rat liver S-9 mix) .

Solubility: Complete solubility of the test substance in DMSO.

Toxicity: A bacteriotoxic effect was observed in the standard plate test at doses => 2500 µg/plate and in the preincubation test at 1500 µg/plate.

Mutagenicity: An increase in the number of his+ revertants was not observed both in the standard plate test and in the preincubation test either without S-9 mix or after the addition of a metabolizing system.

Assessment:

According to the results of the present study, the test substance 2,3,6-Trimethylphenol is not mutagenic in the Ames test under the experimental conditions chosen here.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well documentated study report which meets basic scientific principles.
Reference:
Composition 0
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Test material information:
Composition 1
Target gene:
thymidine kinase (tk) locus
Species / strain:
mouse lymphoma L5178Y cells
Details on mammalian cell lines (if applicable):
- Type and identity of media: Fischer's Media for Leukemic Cells of Mice
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes
Additional strain characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver S9
Test concentrations with justification for top dose:
5.0 to 500 µg/rnL for the non-activated cultures and 1.0 to 150 µg/mL for the S9-activated cultures with a 4-hour exposure
Vehicle:
- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: DMSO was selected as the solvent of choice based on solubility of the test article in study number AB40LW,33 I.BTL and compatibility with the target cells. The test article was soluble in DMSO at approximately 140 mg/mL, the maximum concentration prepared in the preIiminary toxicity test.
Negative controls:
not specified
Solvent controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Migrated to IUCLID6: (MMS), CAS 66-27-3, lot #05713JD, expiration date 15 January 2009, for the non-activated test system at stock concentrations of 1500 and 2000 µg/mL with a 4-hour exposure or 500 and 750 µg/mL with a 24 hour exposure.
Negative controls:
not specified
Solvent controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
Migrated to IUCLID6: (DMBA), CAS 57-97-6, lot # 055K1360, expiration date 20 April 2010 was used at stock concentrations of 75 and 100 µg/mL (initial assay) or 100 and 125 µg/mL (confirmatory assay) as the positive control for the S9-activated test system
Details on test system and conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period:
- Exposure duration: without activation: 4 and 24 hour exposure; with S9 activation: 4 hour exposure
- Expression time (cells in growth medium): 4 houre exposure: 24 and 48 hours after treatment and for 24 hour exposure: 48 and 72 hours after treatment
- Selection time (if incubation with a selection agent): 10 - 14 days
- Fixation time (start of exposure up to fixation or harvest of cells): no data

SELECTION AGENT (mutation assays): trifluorothymidine

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: 1 x 10 e6 cells / plate in triplicate

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:
In evaluation of the data, increases in mutant frequencies which occur only at highly toxic concentrations (i.e., less than 10% total growth) are not considered biologically relevant.
• A result will be considered positive if a concentration-related increase in mutant frequency is observed in the treated cultures and one or more treatment conditions with 10% or greater total growth exhibit mutant frequencies of ≥ 90 mutants per 10e6 clonable cells over the background level (based on the average mutant frequency of duplicate cultures). If the average solvent control mutant frequency is >90 mutants per 10e6 clonable cells, a doubling of mutant frequency over the background will also be required.
• A result will be considered negative if the treated cultures exhibit mutant frequencies of less than 90 mutants per 10e6 clonable cells over the background level (based on the average mutant frequency of duplicate cultures) and there is no concentration-related increase in mutant frequency.
• There are some situations where a chemical may be considered negative when there is no culture showing between 10 - 20% survival:
1) There is no evidence of Mutagenicity (e.g. no dose response or increase in mutant frequencies between 45 and 89 mutants per 10e6 above control) in a series of data points within 100% to 20% survival and there is at least one negative data point between 20% and 25% survival.
2) There is no evidence of Mutagenicity (e.g. no dose response or increase in mutant frequencies between 45 and 89 mutants per 10e6 above control) in a series of data points between 100% to 25% survival and there is also a negative data point between 10% and 1% survival. In this case it is acceptable to count the TFT colonies of cultures exhibiting <10% total growth.
Statistics:
The mutant frequency (number of mutants per 10e6 surviving cells) was determined by dividing the average number of colonies in the TFT plates by the average number of colonies in the corresponding VC plates and multiplying by the dilution factor (2x10e-4) and is expressed as TFT-resistant mutants per 10e6 surviving cells. The induced mutant frequency (IMF) is defined as the mutant frequency of the treated culture minus the mutant frequency of the solvent control cultures. The International Workshop on Genotoxicity established a Global Evaluation Factor (GEF) for a positive response at an IMF of ≥ 90 mutants per 10e6 clonable cells (Moore et al., Mouse Lymphoma Thymidine Kinase GeneMutation Assay: Follow-up Meeting of the International Workshop on Genotoxicity Testing_Aberdeen, Scotland, 2003_Assay Acceptance Criteria, Positive Controls, and Data Evaluation (2006) EMM 47:1-5.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
Substantial toxicity was observed at ≥ 50 µg/mL with and without activation with a 4-hour exposure and without activation with a 24-hour exposure.
Vehicle controls valid:
yes
Negative controls valid:
not specified
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
- A preliminary toxicity assay was used to establish the concentration range for the mutagenesis assays.
- In the preliminary toxicity assay, the maximum concentration of 2,3,6-Trimethylphenol in treatment medium was 1400 µg/rnL (10 mM). No visible precipitate was present at any concentration in treatment medium at the end of treatment Selection of concentrations for the mutation assay was based on reduction of suspension growth relative to the solvent control.
Substantial toxicity, i.e., suspension growth of ≤ 50% of the solvent control, was observed at ≥ 150 g/mL without activation with a 4-hour exposure, ≥ 50 µg/rnL with S9 activation with a 4-hour exposure, and ≥ 150 µg/mL without activation with a 24-hour exposure.

Based on the results of the preliminary toxicity assay, the concentrations tested in the initial mutagenesis assay ranged from 5.0 to 200 µg/mL for the non-activated cultures with a 4-hour and 24 -hour exposure and 1.5 to 75 µg/mL for the S9-activated cultures with a 4-hour exposure.

A supplemental assay (hereinafter referred to as a confirmatory assay) after the receipt of a negative letter from the European Food Safety Authority was performed. A new sample of test article was sent to BioReliance and all phases of the study were repeated.

In the second preliminary toxicity assay, the maximum concentration of 2,3,6-Trimethylphenol in treatment medium was 1400 µg/mL (10 mM). No visible precipitate was present at any concentration in treatment medium at the end of treatment. Selection of concentrations for the mutation assay was based on reduction of suspension growth relative to the solvent control.
Substantial toxicity was observed at ≥ 50 µg/mL with and without activation with a 4-hour exposure and without activation with a 24-hour exposure.

Based on the results of the preliminary toxicity assay, the concentrations tested in the initial mutagenesis assay ranged from 5.0 to 500 µg/mL for the non-activated cultures and 1.0 to 150 µg/mL for the S9-activated cultures with a 4-hour exposure. No visible precipitate was present at any concentration in treatment medium.

Based on the results of the preliminary toxicity assay, the concentrations tested in the extended treatment assay ranged from 2.5 to 200 µg/mL for non-activated cultures with a 24-hour exposure. No visible precipitate was present at any concentration in treatment medium.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
no data
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Test material information:
Composition 1
Target gene:
no target gene
Species / strain:
other: Chinese hamster CHL/IU cells
Details on mammalian cell lines (if applicable):
CHL/IU cells derived from Chinese hamsters were obtained from Dainippon Pharma Co., Ltd (obtained in November 1996, at that time the 14th passage, and at frozen time the 17th passage) were used within the fifth passage after being unfrozen.
Metabolic activation:
with and without
Metabolic activation system:
Rat liver, induced with phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
without S9 mix (24 and 48 hr continuous treatment):
0, 25, 50, 100, 200 µg/ml (main test)
0, 12.5, 25, 50, 100, 200 µg/ml (confirmation test)

without S9 mix (6 hr short-term treatment): 0, 25, 50, 100, 200 µg/ml

with S9 mix (6 hr short-term treatment): 0, 25, 50, 100, 200 µg/ml
Vehicle:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: no data
Negative controls:
not specified
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
other: Mitomycin C (without S9), Benzo[a]pyrene (with S9)
Details on test system and conditions:
Determination of Treatment Concentration in Cell Growth Inhibition Tests
In order to determine treatment concentration of the test substance used in the chromosomal aberration test, influence of the test substance on cell growth was investigated.
Growth inhibition effect with respect to CHL/IU cells by the test substance was measured by means of a densimeter for single layered cultured cells, where the ratio against a negative control group was regarded as an index.
Subsequently, concentration value showing about 50% growth inhibition by 2,3,6-trimethylphenol was calculated by way of probit method. The concentration values were found to be 124 μg/mL and 71 μg/mL with respect to the continuous treatments for 24 hours and 48 hours, respectively. The concentration values showing about 50% growth inhibition in the short time treatments with and without S9 mix were 108 μg/mL and 119 μg/mL, respectively.

Setting of Experimental Groups
From the result of the cell growth inhibition tests, the highest concentration value of the test substance used in chromosomal aberration tests was determined to be 200 μg/mL. Then, four dose values were set with common ratio 2. In confirmation tests with respect to the continuous treatments for 24 hours and 48 hours, the highest concentration value was set to be 200 μg/mL, and five dose values were set with common ratio 2. As positive control substances, 0.03μg/mL MitomycinC and 20 μg/mL benzo[a]pyrene were set for the continuous treatments and short time treatments, respectively.

Method for Producing Chromosome Specimens
Two hours before the end of incubation, colcemid was added to the culture solution so that its final concentration in the culture solution became about 0.1 μg/mL. The chromosome specimens were produced by way of a common procedure. Two slide specimens were produced per dish. The produced specimens were stained by 3% giemsa solution for 20 minutes.

Chromosome Analysis
Among the produced slide specimens, each slide specimen obtained from one dish was analyzed respectively. Here, the treatment conditions encoded so as not to disclose them. The chromosome analysis was performed based on the classification of Mammalian Mutagenicity Study Group (MMS) 1) of the Japanese Environmental Mutagen Society. The existence of structural aberration such as gaps, break and exchange of chromosomes and chromatids as well as the existence of polyploid cells were observed. For the structural aberrations and polyploid, 200 metaphase chromosomes per group were analyzed.
Evaluation criteria:
no data
Statistics:
With respect to the result of the analysis on the solvent and positive control groups, and the treatment groups with the test substance, the number of cells having been observed, types and the number of structural aberrations as well as the number of polyploid cells for each group were summed up and written on a recording sheet. Inducibility of chromosomal aberration by the test substance was determined in accordance with the judgment criterion by Ishidate et al.2) Here, the test substance was determined to be negative if frequency of cells having chromosomal aberration is less than 5%, to be suspected positive if the frequency is 5% or greater and less than 10%, and to be positive if the frequency is 10% or greater.
Species / strain:
other: Chinese hamster (CHL/IU) cells
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity:
yes
Remarks:
124 and 71 ug/ml at 24 and 48h
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: strain/cell type: CHL cells
Remarks:
Migrated from field 'Test system'.
Results on the chromosome analysis by the continuous treatments:
When CHL/IU cells were continuously treated for 24 hours and 48 hours, expression frequencies of structural chromosomal 
aberration were 5.0% with respect to 100 μg/mL after 24 hours treatment, 2.0%, 11.5%, 38.0% and 18.1% with respect 
to 25 μg/mL, 50 μg/mL, 100 μg/mL and 200 μg/mL after 48 hours treatment, respectively. In order to confirm reproducibility 
and dose dependence of the results, confirmation tests were performed by setting 200 μg/mL as the maximum concentration 
and by setting five values of dose with common ratio 2 for the treatments both for 24 hours and 48 hours. The results for the 
treatments after 48 hours were 9.5%, 13.5% and 8.5% for 50 μg/mL, 100 μg/mL and 200 μg/L, respectively.

Results on the chromosome analysis by the short time treatments:
In both short time treatments with S9 mix at 200 μg/mL and without S9 mix at 200 μg/mL, expression frequency of chromosomal 
structural aberration were 11.5% and 11.0%, respectively.
Expression frequency of number of structural aberrations was lower than 5% in all treatment conditions.

Polyploidy was not induced in any treatment group.
Executive summary:

Abstract

We conducted chromosomal aberration tests using cultured Chinese hamster cells (CHL/IU) in order to study an cytogenetic influence of 2,3,6-trimethylphenol on the cultivated cells.

Based on the result of cell growth inhibition test, 200 μg/mL was set to be the maximum concentration, and four concentration values with common ratio 2 were set with respect to continuous treatments for 24 hours and 48 hours as well as with respect to short time treatments with and without S9 mix, respectively.

As the result of the continuous treatments of CHL/IU cells for 24 hours and 48 hours, expression frequency of chromosomal structural aberration was 5.0% in the case of 100μg/mL after treatment for 24 hours. It was 2.0%, 11.5%, 38.0% and 18.1% for 25 μg/mL, 50 μg/mL, 100 μg/mL and 200 μg/mL after treatment for 48 hours, respectively. In order to confirm reproducibility and dose dependence of the result, confirmation tests were conducted by setting 200 μg/mL as the maximum concentration and by setting five values of dose with common ratio 2 for treatments both for 24 hours and 48 hours. The results for the treatment for 48 hours were 9.5%, 13.5% and 8.5% for 50 μg/mL, 100 μg/mL and 200 μg/L, respectively.

In both the short time treatments with S9 mix with respect to 200 μg/mL and the treatments without S9 mix with respect to 200 μg/mL, expression frequency of chromosomal structural aberration were 11.5% and 11.0%, respectively.

Expression frequency of numerical aberration was lower than 5% in all treatment conditions.

Based on the above-mentioned results, it is concluded that 2,3,6-trimethylphenol induces chromosomal aberration (positive) under the conditions of this test system.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

in vivo studies
Micronucleus assay, mouse: negative (GLP, OECD 474, BASF 2003)

Link to relevant study records
Reference
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
Reference:
Composition 0
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 information:
Composition 1
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)
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
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
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
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls valid:
yes
Positive controls valid:
yes

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.

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.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

in vitro studies

 

Gene mutation in bacteria 

In a bacterial reverse gene mutation assay according to OECD 471 (BASF, 1989), S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 were exposed to 2,3,6-Trimethylphenol (CAS No. 2416-94-6) in DMSO at concentrations of 4, 20, 100, 500, 1500, 2500 and 5000 µg/plate in the presence and absence of mammalian metabolic activation (Standard Plate Test and Preincubation Test). 2,3,6-Trimethylphenol induced no increase in the number of revertants in any tester strain under any test condition. Cytotoxicity (indicated by reduced bacterial growth) was observed in the Standard Plate Test at doses ≥ 2500 µg/plate and in the Preincubation Test at doses ≥ 1500 µg/plate.

In a further bacterial reverse gene mutation assay according to OECD 471 and 472 (Miyagawa et al, 1999), S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A were exposed to 2,3,6-Trimethylphenol (CAS No. 2416-94-6) in DMSO at concentrations of 39, 78, 156, 313, 625, and 1250 µg/plate in the presence and absence of mammalian metabolic activation (Preincubation Test). The number of reverse mutated colonies induced by the test substance at each concentration value did not exceed, independent of the existence of S9 mix, twice or more with respect to either strain compared to negative control value. Dependent on the strain, cytotoxicity was observed with and without S9 mix at doses ≥ 625 µg/plate. 

 

Gene mutation in mammalian cells 

In an in vitro mammalian cell gene mutation test according to OECD 476 (BioReliance 2008), mouse lymphoma L5178Y cells were treated with 2,3,6 -trimethylphenol (CAS No. 2416-94 -6) in DMSO at concentrations of 5.0 to 500 µg/mL without metabolic activation and at 1.0 to 150 µg/mL with metabolic activation with a 4- and 24 hour exposure. Substantial toxicity was observed at50 µg/mL with and without activation with a 4-hour exposure and without activation with a 24-hour exposure. No genotoxic effects were measured.

 

Cytogenicity in mammalian cells 

In a mammalian cell cytogenetics assay [Chromosome aberration Test] according to OECD 473 (Ohta et al, 1999), CHL/IU cell cultures were exposed to 2,3,6 -trimethylphenol (CAS No. 2416-94-6) in acetone at concentrations of 0 – 200 µg/ml with and without metabolic activation with phenobarbital- and benzoflavone-induced rat liver S-9 Mix. 2,3,6-Trimethylphenol was tested up to cytotoxic concentrations. When CHL/IU cells were continuously treated for 24 hours and 48 hours, expression frequencies of structural chromosomal aberration were 5.0% with respect to 100μg/ml after 24 hours treatment, 2.0%, 11.5%, 38.0% and 18.1% with respect to 25μg/ml, 50μg/ml, 100μg/ml and 200μg/ml after 48 hours treatment, respectively. In short time (6-hour) treatments with S9 mix at 200μg/ml and without S9 mix at 200μg/ml, expression frequency of chromosomal structural aberration were 11.5% and 11.0%, respectively. Expression frequency of number of structural aberrations was lower than 5% in all treatment conditions. Polyploidy was not induced under any conditions. Positive controls induced the appropriate response.

in vivo studies

In a NMRI mouse bone marrow micronucleus assay (BASF, 2003), 6 males/dose were treated by intraperitoneal injection with 2,3,6 -Trimethylphenol (CAS No. 2416-94-6) at doses of 0, 100, 200, 300 mg/kg bw. Bone marrow cells were harvested at 24 and 48 hours post-treatment. The vehicle was olive oil (acute dosing).There were evident signs of toxicity during the study. 2,3,6 -trimethylphenol was tested at an adequate dose. The positive control induced the appropriate response.


Justification for classification or non-classification

Based on reliable studies performed with the test compound 2,3,6 -trimethylphenol, classification regarding genetic toxicity according to EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008 is not warranted.