2,4,6-trimethylphenol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation study in bacteria (OECD TG 471), 2022: negative in all five tested strains with and without metabolic activation
In vitro gene mutation study in mammalian cells (OECD TG 476), 2002: equivocal in mouse lymphoma L5178Y cells without metabolic activation, negative with metabolic activation
In vitro cytogenicity / chromosome aberration study in mammalian cells (OECD TG 473), 2002: positive in Chinese hamster ovary (CHO) cells with metabolic activation, negative without metabolic activation

 

 

Link to relevant study records

Reference

Reference 1

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant study, available as unpublished report, no restrictions, fully adequate for assessment.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

- Preliminary toxicity assay: nine concentrations between 0.136, 0.408, 1.36, 4.08, 13.6, 40.8, 136, 408, 1360 µg/mL
- With metabolic activation, 4 h exposure: 12.5, 25, 50, 100, 150, 200 µg/mL
- Without metabolic activation, 4 h exposure: 25, 50, 100, 150, 200, 300 µg/mL
- Without metabolic activation, 20 h exposure: 25, 50, 100, 150, 200, 300, 400 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO was determined to be the solvent of choice based on the solubility of the test article and compatibility with the target cells

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

0.1 and 0.2 µg/mL mitomycin without metabolic activation; 10 and 20 µg/mL cyclophosphamide with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION:
- Exposure duration: without metabolic activation 4 and 20 hours, with metabolic activation 4 hours
- Expression time (cells in growth medium): 20 hours

SPINDLE INHIBITOR (cytogenetic assays): Two hours prior to harverst Colcemid was added to duplicate flasks for each treatment condition.

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: The percentage of cells in mitosis per 500 cells scored (mitotic index) was determined for each treatment group. Metaphase cells with 20 ± 2 centromeres were examined under oil immersion without prior knowledge of treatment groups. A minimum of 200 metaphase spreads were examined and scored for chromate-type and chromosome-type aberrations. Chromatid-type aberrations include chromatid and isochromatid breaks and exchanges figures such as quadriradials (symmetrical and asymmetrical interchanges), triradials, and complex rearrangements. Chromosome-type aberrations includes chromosome breaks and exchanges figures such as dicentrics and rings.

DETERMINATION OF CYTOTOXICITY:
- Method: relative total growth and mitotix index
- Tryptan blue dye exclusion was used to determine viability

STAINING: Slides were stained with 5% Giemsa

Evaluation criteria:

- Selection of doses for microscopic analysis was based on toxicity (the lowest dose with at least 50% reduction in cell growth relative to the solvent control and two lower doses) in the non-activated 4 hour exposure group. Selection of doses for microscopic analysis was based on mitotic inhibition (the lowest dose with at least 50% reduction in mitotic index relative to the solvent control and two lower doses) in the S9 activated 4 hour exposure group and in the non-activated 20 hour exposure group.
- Validity criteria: The frequency of cells with structural chromosome aberrations in the solvent must be within the range of the historical solvent control. The percentage of cells with chromosome aberrations in the positive control must be statistically increased (p≤0.05, Fischer's exact test) relative to solvent control.
- The test article was considered to induce a positive response when the percentage of cells with aberrations is increased in a dose-responsive manner with one or more concentrations being statistically significant (p≤0.05). However, values that are statistically significant but do not exceed the range of historic solvent controls may be judged as not biologically significant. Test articles not demonstrating a statistically significant increase in aberrations will be concluded to be negative.

Statistics:

Fisher's exact test was used to compare pairwise the percent aberrant cells of each treatment group with that of the solvent control. In the event of a positive Fisher's test at any test article dose level, the Cochran-Armitage test was used to measure dose-responsiveness.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

structural aberrations

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: the pH of the highest concentration of test article in treatment medium was approximately 6.5.
- Effects of osmolality: The osmolality in treatment medium of the highest concentration tested, 400 µg/mL, was 386 mmol/kg. The osmolality of the solvent in treatment medium was 426 mmol/kg.
- Precipitation: No precipitation was observed in the main assay.

RANGE-FINDING/SCREENING STUDIES:
Visible precipitate was observed in treatment medium at dose level 1360 µg/mL. Dose levels ≤408 µg/mL were soluble in treatment medium. The osmolality in treatment medium of the highest soulble concentration, 408 µg/mL, was 384 mmol/kg. The osmolality of the solvent in treatment medium was 424 mmol/kg. The pH of the highest concentration of test article in treatment medium was approximately 6.5. Substantial toxicity (i.e. 50% of growth inhibition) was observed at dose levels ≥ 408 µg/mL in 4 hour exposure group without metabolic activation, at dose levels ≥ 136 µg/mL in the 4 hour exposure group with metabolic activation and in the 20 hour exposure group without metabolic activation.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Toxicity of the test substance in CHO cells when treated for 4 hours in the absence of S9 activation was 63% at 300 µg/mL, the highest test concentration evaluated. The mitotic index at the highest dose level evaluated for chromosome aberrations, 300µg/mL, was 40% reduced relative to the solvent control. The dose levels selected for microscopic analysis were 50, 150, and 300 µg/mL. Toxicity of the test substance in CHO cells when treated for 4 hours in the presence of S9 activation was 35% at 200 µg/mL, the highest test concentration evaluated. The mitotic index at the highest dose level evaluated for chromosome aberrations, 200µg/mL, was 61% reduced relative to the solvent control. The dose levels selected for microscopic analysis were 50, 100, and 200 µg/mL. Toxicity of the test substance was 40% at 100 µg/mL, the highest test concentration evaluated in the non-activated 20 hour continuous exposure group, the highest test concentration evaluated. The mitotic index at the highest dose level evaluated for chromosome aberrations, 100 µg/mL, was 56% reduced relative to the solvent control. The dose levels selected for microscopic analysis were 25, 50, and 100 µg/mL.

COMPARISON WITH HISTORICAL CONTROL DATA:
The percentage of cells with structural aberrations in CHO cells when treated for 4 hours in the absence of S9 activation was significantly increased above that of the solvent control at dose level 300 µg/mL (p≤0.05, Fischer's test). The Cochran-Armitage test was also positive for a dose-response (p<0.05). However, the percentage of cells with structural aberrations in the treated group (3.0%) was within the historical solvent control range of 0 - 5.5%. Therefore, it is not considered to be biologically significant.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
positive with metabolic activation
negative without metabolic activation

The test substance is considered to be mutagenic in the presence of metabolic activation in this chromosomal aberration test.

Executive summary:

In a GLP-compliant chromosome aberration test Chinese hamster ovary (CHO) cells were exposed to the test substance in DMSO with and without metabolic activation (S9 -mix). The cells were treated for 4 and 20 hours in the non-activated test system and for 4 hours in the S9 activated test system and all cells were harvested at 20 hours after treatment initiation. Different dosages (up to 200, 300 and 400 µg/mL, for +S9 mix 4 h, -S9 mix 4 h and -S9 mix 20 h, respectively) were chosen in the three groups. Cytotoxicity was observed in all three groups at the highest tested dose. The percentage of cells with structural aberrations in the test article-treated groups was significantly increased above that of the solvent control at dose levels 100 and 200 µg/mL in the S9 activated 4 hour exposure group. The precentage of numerical aberrations was not significantly increased. In the non-activated 4 -hour exposure group, the percentage of cells with structural aberrations was significantly above that of the solvent control at dose level 300 µg/mL. However, this percentage of 3.0 % was within the historical solvent control and therefore not considered to be biologically relevant. In the absence of the S9 mix in the 20 -hour exposure group, the test article did not increase the frequency of cells with aberrations. Based on these results the test substance is considered to be mutagenic in the presence of metabolic activation.

Reference 2

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:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant study, available as unpublished report, no restrictions, fully adequate for assessment.

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

- Preliminary toxicity assay: nine concentrations between 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1360 µg/mL
- With metabolic activation: 10, 30, 40, 50, 100 µg/mL
- Without metabolic activation: 50, 75, 100, 125, 150 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO was determined to be the solvent of choice based on the solubility of the test article and compatibility with the target cells.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

2.5, 5, 10, 20 µg/mL

Positive control substance:

7,12-dimethylbenzanthracene

methylmethanesulfonate

Remarks:

2.5, 5, 10, and 20 µg/mL methylmethanesulfonate without metabolic activation; 5 and 7.5 µg/mL 7,12-dimethylbenzanthracene with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: without metabolic activation 4 or 24 hours; with metabolic activation 4 hours
- Expression time (cells in growth medium): 48 hours after initial exposure
- Selection time (if incubation with a selection agent): 10 - 14 days

SELECTION AGENT (mutation assays): trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: suspension growth

Evaluation criteria:

- A result was considered positive if a concentration-related increase in mutant frequency was observed and one or more dose levels with 10% or greater total growth exhibited mutant frequencies of ≥100 mutant per 1E6 clonable cells over the background level.
- A result was considered equivocal in the mutant frequency in treated cultures was between 55 and 99 mutants per 1E6 clonable cells over the background level.
- A result was considered negative if the mutant frequency in treated cultures was fewer than 55 mutants per 1E6 clonable cells over the background level.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No visible precipitation was present at any dose level.

RANGE-FINDING/SCREENING STUDIES:
Visible precipitation was observed in treatment medium at dose level 1360 µg/mL. No visible precipitate was present at concentration ≤500 µg/mL in treatment medium Suspension growth relative to the solvent controls was 0% at 500 µg/mL without activation with 4- and 24-hour exposures and 0% at 150 µg/mL with S9 activation.

ADDITIONAL INFORMATION ON GENOTOXICITY:
- Without metabolic activation, 4 hour exposure: One cloned culture (125 µg/L) exhibited a mutant frequency of 100 mutants per 1E6 clonable cells over that of the solvent control, and five cultures (75, 100, 125, and 150 µg/L) exhibited mutant frequencies that were between 55 and 99 mutants per 1E6 clonable cells over that of solvent control. There was no dose-response trend.
- With metabolic activation, 4 hour exposure: One cloned culture (40 µg/L) exhibited a mutant frequency that was between 55 and 99 mutants per 1E6 clonable cells over that of solvent control. There was no dose-response trend.
- Without metabolic activation, 24 hour exposure: One cloned culture (100µg/mL) exhibited a mutant frequency of 115 mutants per 1E6 clonable cells over that of the solvent control, and two cloned cultures (75 and 100 µg/mL) exhibited mutant frequencies between 55 and 99 mutants per 1E6 clonable cells over that of solvent control. There was no dose-response trend.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Suspension growth of ≤50% of the solvent control, was observed at ≥75 µg/mL without activation with 4-hour exposure and ≥30 µg/mL with activation with a 4-hour exposure. Toxicity in cloned cultures was observed at doses 150 µg/mL without activation with 24-hour exposure.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
ambiguous without metabolic activation
negative with metabolic activation

Under the conditions of this study, the test substance was concluded to be equivocal without activation and negative with S9 activation for mutagenicity in mammalian cells.

Executive summary:

In a GLP compliant in vitro Mouse Lymphoma Assay, L5178Y cells were exposed to the test substance with and without metabolic activation (S9 mix) to investigate the potential of the test substance to induce gene mutations. The cells were treated for 4 and 24 hours in the non-activated test system, and for 4 hours in the S9 activated test system. Different dosages (up to 100 and 150 µg/mL, with and without metabolic activation, respectively) were chosen. Cytotoxicity (total growth ≤50% of the solvent control) was observed at ≥75 and 150 µg/mL without activation with 4-hour and 24 -hour exposure, respectively, and at dose levels ≥30 µg/mL with metabolic activation. In the presence of S9 mix the test substance induced in one culture (40 µg/mL) a mutant frequency between 55 and 99 over solvent control. All other mutant frequencies were below 55 over solvent control and therefore the results were negative in the presence of S9 -mix. In the absence of S9 mix results were equivocal, because in both the 4 -hour and 24 -hour treatment one cultures exhibited a mutant frequency of ≥100 mutants per 1E6 clonable cells over that of the solvent control. However, no dose-response was observed, but as for both exposure times several cultures exhibited mutant frequencies between 55 and 99 mutants per 1E6 clonable cells over that of the solvent control, the results in the absence of a metabolic activation system were considered to be equivocal.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 Sept - 5 Nov 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted in 2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

hisC3076 (for S. typhimurium strain TA1537)
hisD3052 (for S. typhimurium strain TA98)
hisG46 (for S. typhimurium strain TA100 and TA1535)
hisG428 (for S. typhimurium strain TA102)

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Trinova Biochem GmbH, Gießen.
- method of preparation of S9 mix: Produced from the livers of male Sprague-Dawley rats which
were treated with Phenobarbital/5,6-Benzoflavone
- Volume of S9 mix in the final culture medium: 500 µL

Test concentrations with justification for top dose:

Experiment 1 (plate incorporation method):
50, 150, 500, 1500, 5000 µg/plate
Experiment 1b (plate incorporation method):
197.5, 296.3, 444.4, 666.7, 1000.0, 1500.0 µg/plate
Experiment 2 (pre-incubation method):
20.1, 61.7, 185.2, 555.6, 1666.7, 5000.0 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: Based on the non-GLP pre-test, DMSO was chosen as vehicle, because the test item was sufficiently soluble, and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO and water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

mitomycin C

other:

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): 3
- Number of independent experiments: 3

METHOD OF TREATMENT/ EXPOSURE:

- Test substance added in agar for plate incorporation test (Experiment 1 and 1b) and in bacterial suspension for pre-incubation (Experiment 2).

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: incubated at 37 ± 1 °C for 20 minutes
- Exposure duration/duration of treatment: 48 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

METHODS FOR MEASUREMENTS OF GENOTOXICIY
A result is considered as clearly positive if all following criteria are fulfilled:
- a concentration-related increase, in revertants
- a clear biological relevant increase in at least one concentration compared to the concurrent solvent control
- at least one concentration with an increase above the distribution of historical solvent control data (mean ± 3 SD).

Evaluation criteria:

A biologically relevant increase is described as:
- if in the bacteria strains TA98, TA100, TA102 the number of revertants is at least twice as high than the reversion rate of the negative controls (increase factor of at least 2.0)
- if in the bacteria strains TA1535 and TA1537 the number of revertants is at least three times higher than the reversion rate of the negative controls (increase factor of at least 3.0).
A test result is considered as clearly negative, if it does not meet these criteria.

Statistics:

Means and standard deviation of triplicate plates were calculated.

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At a test item concentration of 5000 µg/plate the bacterial background lawn was absent. At a test item concentration of 1500 µg/plate, the bacterial background lawn was reduced for TA1537 (-/+S9).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535 pSK1002

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Treated up to 1500 µg/plate. At a test item concentration of 1500 µg/plate, the bacterial background lawn was reduced for TA1535 (-S9) and the number of revertants was clearly reduced for TA1535 (-S9).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Treated up to 1500 µg/plate. At a test item concentration of 1500 µg/plate, the bacterial background lawn was reduced for TA102 (-S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Treated up to 1500 µg/plate. At a test item concentration of 5000 µg/plate the bacterial background lawn was absent.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 1666.7 µg/plate no colonies were found for TA98 (-/+S9). At a test item concentration of 5000 µg/plate the bacterial background lawn was absent for all tested strains and no revertant colonies were found (except from 1 colony for TA98 (-S9)).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

STUDY RESULTS
All strains met the criterion of at least 109 bacteria/mL (correlating to 100 colonies/plate after dilution), and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the vehicle and negative controls were in the normal range of the test laboratory (mean ± 3 standard deviations). All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and all were within the historical control data ranges.

Experiment 1:
The test item showed no precipitates on the plates in all tested concentrations. At a test item concentration of 5000 µg/plate the bacterial background lawn was absent for all tested strains and no revertant colonies were found (except from 1 colony for TA98 (-S9)). At a test item concentration of 1500 µg/plate, the bacterial background lawn was reduced for TA98 (-S9), TA100 (-S9), TA102 (-S9), TA1535 (-S9) and TA1537 (-/+S9), no colonies were found for TA100 (-S9) and TA1537 (-/+S9) and the number of revertants was clearly reduced for TA98 (-S9), TA100 (+S9), TA102 (-S9), TA1535 (-S9). Thus, signs of toxicity towards the bacteria strains and in the concentrations described above could be observed. In the lower concentrations, no signs of cytotoxicity were found. No relevant or concentration-related increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. Based on the toxicity results and to verify this result, a further experiment was performed with adapted concentrations (Exp. 1b). The mean revertant values of Experiment 1 are shown in Table 1

Experiment 1b:
The test item showed no precipitates on the plates in all tested concentrations. At a test item concentration of 1500 µg/plate, the bacterial background lawn was reduced for TA100 (-/+S9) and TA1537 (-/+S9), no colonies were found for TA100 (-/+S9) and TA1537 (-/+S9) and the number of revertants was clearly reduced for TA98 (-/+S9), TA102 (-S9), TA1535 (-S9). At a test item concentration of 1000.0 µg/plate, the bacterial background lawn was visible and not affected, but no colonies were found for TA1537 (-S9) and a clear reduction of colony numbers was observed for TA102 (-S9) and TA1537 (+S9). At 666.7 µg/plate the number of revertants was still reduced for TA1537 (-/+S9) and for TA1537 (+S9) also at 444.4 µg/plate. Thus, signs of toxicity towards the bacteria strains and in the concentrations described above could be observed. In the lower concentrations, no signs of cytotoxicity were found. No relevant or concentration-related increase of the number of revertant colonies in all treated strains at all concentrations tested with and without metabolic activation could be observed. To verify this result, a further experiment with adapted conditions (pre-incubation method) was performed (Experiment 2). The mean revertant values of Experiment 1b are shown in Table 2 under "Any other information on results incl. tables".

Experiment 2:
The test item showed no precipitates on the plates in all tested concentrations. At a test item concentration of 5000 µg/plate the bacterial background lawn was absent for all tested strains and no revertant colonies were found. At a test item concentration of 1666.7 µg/plate, the bacterial background lawn was reduced for all tested strains, no colonies were found for TA98 (-/+S9), TA100 (-/+S9), TA102 (-/+S9) and TA1537 (-S9) and a clear reduction in colony numbers was observed for TA1535 (-/+S9) and TA1537 (+S9). Thus, signs of toxicity towards the bacteria strains and in the concentrations described above could be observed. In the lower concentrations, no signs of cytotoxicity were found. No relevant or concentration-related increase of the number of revertant colonies in all treated strains at all concentrations tested with and without metabolic activation could be observed.
The mean revertant values of Experiment 2 are shown in Table 3 under "Any other information on results incl. tables".

The study was performed with the plate incorporation (Exp. 1 and 1b) and pre-incubation method (exp. 2) in the absence and presence of a metabolic activation system (S9). Under these conditions the influence of the test item on bacterial test strains was evaluated. The test item 2,4,6-trimethylphenol showed no relevant or dose-related increase in the number of revertants in the Salmonella typhimurium test strains TA98, TA100, TA102, TA1535 and TA1537 in all evaluated experiments. Based on the results of this study it is concluded that 2,4,6-trimethylphenol is not mutagenic in the Salmonella typhimurium test strains TA98, TA100, TA102, TA1535 and TA1537 in the absence and presence of metabolic activation under the experimental conditions of the study.


The historical data of the laboratory are shown in Table 4 under "Any other information on results incl. tables". The results of the Experimental data fall within the historical control data range

Table 1 Mean Revertants Experiment 1 - Plate incorporation

Strain		TA98		TA100		TA102		TA1535		TA1537
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. water	Mean	16	14	54	57	153	148	10	8	7	7
	sd	3.2	1.7	3.0	2.5	6.1	0.0	0.6	0.6	1.7	1.0
DMSO	Mean	13	15	55	55	148	160	9	8	5	6
	sd	1.0	2.6	0.6	3.1	4.0	6.9	1.5	0.6	1.5	1.2
Positive Controls*	Mean	405	88	347	1443	728	883	203	127	63	94
	sd	28.1	12.0	12.2	16.7	34.9	48.2	8.3	6.1	1.5	6.7
	f(I)	31.15	5.87	6.43	26.24	4.76	5.52	20.30	15.88	12.60	15.67
5000 µg/plate	Mean	0	0	0	0	0	0	0	0	0	0
	sd	0.6	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
	f(I)	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
1500 µg/plate	Mean	3	8	0	26	51	153	1	7	0	0
	sd	0.6	2.1	0.0	6.1	9.0	4.6	0.6	1.0	0.0	0.0
	f(I)	0.23	0.53	0.00	0.47	0.34	0.96	0.11	0.88	0.00	0.00
500 µg/plate	Mean	10	11	58	56	157	163	7	7	4	4
	sd	1.7	2.0	5.5	3.5	12.2	6.1	1.7	1.0	1.2	1.5
	f(I)	0.77	0.73	1.05	1.02	1.06	1.02	0.78	0.88	0.80	0.67
150 µg/plate	Mean	13	16	51	55	155	159	7	8	5	5
	sd	1.2	1.7	4.2	4.6	6.1	9.2	1.0	2.0	0.6	1.0
	f(I)	1.00	1.07	0.93	1.00	1.05	0.99	0.78	1.00	1.00	0.83
50 µg/plate	Mean	13	15	60	54	153	156	6	7	4	5
	sd	1.2	1.5	4.5	4.2	12.2	4.0	0.6	0.6	0.6	1.5
	f(I)	1.00	1.00	1.09	0.98	1.03	0.98	0.67	0.88	0.80	0.83

sd = standard deviation ±
* Different positive controls were used

f(I)= increase factor, calculation 
bold marked values = relevant increase in the number of revertants
italic marked values = cytotoxicity

 

Table 2 Mean Revertants Experiment 1b - plate incorporation

Strain		TA98		TA100		TA102		TA1535		TA1537
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. water	Mean	12	14	53	50	167	--	7	--	3	6
	sd	1.5	2.1	11.3	2.5	8.3	--	1.5	--	1.0	2.1
DMSO	Mean	12	15	55	48	172	--	7	--	3	5
	sd	1.7	1.7	3.1	8.2	4.0	--	1.5	--	0.6	1.5
Positive Controls*	Mean	389	87	317	1760	553	--	328	--	67	168
	sd	18.5	8.3	22.3	216.0	14.0	--	13.9	--	4.5	12.0
	f(I)	32.42	5.80	5.98	36.67	3.31	--	46.86	--	22.33	33.60
1500.0 µg/plate	Mean	1	6	0	0	23	--	2	--	0	0
	sd	1.2	1.2	0.0	0.0	3.2	--	1.5	--	0.0	0.0
	f(I)	0.08	0.40	0.00	0.00	0.13	--	0.29	--	0.00	0.00
1000.0 µg/plate	Mean	6	11	37	55	84	--	5	--	0	1
	sd	1.5	1.2	6.1	3.0	12.0	--	0.6	--	0.0	1.0
	f(I)	0.50	0.73	0.67	1.15	0.49	--	0.71	--	0.00	0.20
666.7 µg/plate	Mean	7	13	53	52	165	--	7	--	1	2
	sd	1.0	3.1	5.2	1.0	12.2	--	1.5	--	0.6	1.7
	f(I)	0.58	0.87	0.96	1.08	0.96	--	1.00	--	0.33	0.40
444.4 µg/plate	Mean	12	15	50	59	179	--	6	--	2	1
	sd	2.9	1.7	9.1	6.1	2.3	--	1.2	--	1.2	0.6
	f(I)	1.00	1.00	0.91	1.23	1.04	--	0.86	--	0.67	0.20
296.3 µg/plate	Mean	10	14	54	60	181	--	7	--	3	4
	sd	1.5	4.7	9.1	12.5	4.6	--	1.2	--	1.0	2.5
	f(I)	0.83	0.93	0.98	1.25	1.05	--	1.00	--	1.00	0.80
197.5 µg/plate	Mean	12	18	55	56	175	--	8	--	2	7
	sd	2.1	1.0	6.1	14.1	8.3	--	1.2	--	1.5	1.5
	f(I)	1.00	1.20	1.00	1.17	1.02	--	1.14	--	0.67	1.40

sd = standard deviation ±
* Different positive controls were used 

f(I)= increase factor, calculation 
bold marked values = relevant increase in the number of revertants
italic marked values = cytotoxicity

-- = not tested

 

Table 3       Mean Revertants Experiment 2 - pre-incubation

 

Strain		TA98		TA100		TA102		TA1535		TA1537
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. water	Mean	11	14	53	52	153	147	8	7	4	5
	sd	2.1	2.1	4.0	4.0	4.6	4.6	1.5	2.1	1.5	1.0
DMSO	Mean	12	16	51	53	147	153	8	7	3	4
	sd	0.6	2.3	2.1	1.0	6.1	6.1	2.1	1.7	0.6	0.6
Positive Controls*	Mean	416	116	225	1280	739	619	269	88	62	141
	sd	28.8	18.3	26.0	76.3	37.8	28.1	24.4	6.9	6.0	18.0
	f(I)	34.67	7.25	4.25	24.15	4.83	4.05	33.63	12.57	20.67	35.25
5000 µg/plate	Mean	0	0	0	0	0	0	0	0	0	0
	sd	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
	f(I)	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
1666.7 µg/plate	Mean	0	0	0	0	0	0	1	1	0	0
	sd	0.0	0.0	0.0	0.0	0.0	0.0	1.2	2.3	0.0	0.6
	f(I)	0.00	0.00	0.00	0.00	0.00	0.00	0.13	0.14	0.00	0.00
555.6 µg/plate	Mean	6	9	40	52	125	159	5	7	3	3
	sd	1.0	1.2	7.4	7.9	12.9	6.1	2.3	1.2	1.0	0.0
	f(I)	0.50	0.56	0.78	0.98	0.85	1.04	0.63	1.00	1.00	0.75
185.2 µg/plate	Mean	10	16	54	51	139	137	5	7	4	6
	sd	0.0	2.6	2.0	1.7	11.5	8.3	0.6	1.2	1.0	0.6
	f(I)	0.83	1.00	1.06	0.96	0.95	0.90	0.63	1.00	1.33	1.50
61.7 µg/plate	Mean	11	12	52	53	149	151	7	7	4	4
	sd	1.5	1.5	2.3	4.6	6.1	4.6	2.0	1.0	1.0	1.2
	f(I)	0.92	0.75	1.02	1.00	1.01	0.99	0.88	1.00	1.33	1.00
20.6 µg/plate	Mean	11	15	53	50	153	149	7	9	4	5
	sd	0.0	1.0	4.6	1.0	8.3	4.6	1.7	2.3	0.6	1.2
	f(I)	0.92	0.94	1.04	0.94	1.04	0.97	0.88	1.29	1.33	1.25

sd = standard deviation ±

* Different positive controls were used 

f(I)= increase factor, calculation 

bold marked values = relevant increase in the number of revertants

italic marked values = cytotoxicity

 

Table 4  Historical Data of Spontaneous Revertants

 

Strain		TA98		TA100		TA102		TA1535		TA1537
Induc- tion		- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9
Demin. water	Mean	16	20	67	68	202	201	8	8	5	5
	Min	10	12	46	44	143	137	4	4	3	3
	Max	38	38	100	101	385	399	14	13	7	8
	sd	5	5	13	13	49	51	2	2	1	1
	Range mean ± 2 sd	6-27	10-29	40-94	41-95	104- 300	100- 302	5-12	5-12	3-7	3-7
	Range mean ± 3 sd	0-33	5-34	27- 108	28- 109	55- 349	49- 353	3-14	3-13	2-8	2-8
	Exp 1	16	14	54	57	153	148	10	8	7	7
	Exp. 1b	12	14	53	50	167	--	7	--	3	6
	Exp 2	11	14	53	52	153	147	8	7	4	5
DMSO	Mean	16	19	65	68	209	204	8	8	4	5
	Min	7	8	42	39	141	149	5	5	3	3
	Max	33	38	101	102	483	409	14	15	6	7
	sd	5	6	14	14	64	50	2	2	1	1
	Range mean ± 2 sd	5-26	8-31	38-92	40-95	80- 337	103- 305	5-12	5-12	2-6	3-6
	Range mean ± 3 sd	0-31	3-36	24- 106	27- 108	16- 401	53- 355	3-14	3-14	1-7	2-7
	Exp 1	13	15	55	55	148	160	9	8	5	6
	Exp. 1b	12	15	55	48	172	--	7	--	3	5
	Exp 2	12	16	51	53	147	153	8	7	3	4
Posi- tive Con- trols*1	Mean	613	137	457	1328	807	817	272	159	83	93
	Min	121	62	261	717	481	451	155	59	33	33
	Max	1288	675	696	2389	1917	1613	495	297	243	232
	sd	223	85	115	361	290	269	79	53	35	44
	Range mean ± 2 sd	167- 1059	0*- 308	226- 688	606- 2050	227- 1386	280- 1354	114- 430	53- 266	12- 153	5-182
	Range mean ± 3 sd	0*- 1282	0*- 393	111- 803	245- 2411	0*- 1676	11- 1622	35- 509	0-319	0*- 189	0*- 226
	Exp 1	405	88	347	1443	728	883	203	127	63	94
	Exp. 1b	389	87	317	1760	553	--	328	--	67	168
	Exp 2	416	116	225	1280	739	619	269	88	62	141

sd = standard deviation ±

* Calculated values are < 0. Since these values have no biological relevance, they are set equal to 0

*¹ Different positive controls were used, 

-- = not tested

 

 

Conclusions:

In this Reverse Mutation Assay using strains of Salmonella typhimurium (OECD TG 471) the test item did not induce an increase in the frequency of revertant colonies that met the criteria for a positive result, either with or without metabolic activation (S9-mix). Under the conditions of this test, the test item was considered to be non-mutagenic.

Executive summary:

This study was performed in order to evaluate the mutagenic potential of 2,4,6-trimethylphenol in the Bacterial Reverse Mutation Test using five strains of Salmonella typhimurium (TA98, TA100, TA102, TA1535 and TA1537) based on the most recent Guidelines OECD 471 (2020) and EU Method B.13/14 (2008). The test was performed in three valid experiments in the presence and absence of metabolic activation, with +S9 standing for the presence of a metabolic activation, and -S9 standing
for absence of metabolic activation. In the first experiment, the test item (dissolved in Dimethyl sulfoxide, DMSO) was tested up to concentrations of 5000 µg/plate in the absence and presence of S9 mix in the strains TA98, TA100, TA102, TA1535 and TA1537 using the plate incorporation method. The test item showed no precipitates on the plates at any of the concentrations. Cytotoxicity could be observed in the presence and the absence of metabolic activation in all tested strains. The results of this experiment showed that none of the tested concentrations induced a relevant or concentration-related increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation. Based on the toxicity results of the Experiment 1, the experiment was repeated under the
same conditions with adapted concentrations for the strains TA98 (-/+S9), TA100 (-/+S9),
TA102 (-S9), TA1535 (-S9) and TA1537 (-/+S9). The test item (dissolved in DMSO) was tested up to concentrations of 1500 µg/plate in the presence and absence of metabolic activation. The test item showed no precipitates on the plates at any of the concentrations. Cytotoxicity could be observed in the presence and the absence of metabolic activation in all tested strains. The results of this experiment showed that none of the tested concentrations induced a relevant or concentration-related increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation. 
Based on the results of the Exp. 1 and 1b, the test item was tested up to concentrations of
5000 µg/plate in the presence and absence of S9 mix in all bacteria strains using the preincubation method. The test item showed no precipitates on the plates at any of the concentrations. Cytotoxicity
could be observed in the presence and the absence of metabolic activation in all tested strains. The results of this experiment showed that none of the tested concentrations induced a relevant or concentration-related increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.
Based on the results of this study it is concluded that 2,4,6-trimethylphenol is not mutagenic in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 in the presence and absence of metabolic activation under the experimental conditions in this study.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus (OECD TG 474), 2002: negative (3 concentrations (125, 250 or 500 mg/kg bw) administered by intraperitoneal injection to mice)

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant study, available as unpublished report, no restrictions, fully adequate for assessment.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

ICR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Sprague Dawley, Inc., Frederick, MD
- Age at study initiation: 6-8 weeks
- Weight at study initiation: male 24.7 – 32.2 g, female 24.7 – 29.2 g
- Housing: five per cage per sex in polycarbonate cages which were maintained on stainless steel racks equipped with automatic watering manifolds and which were covered with filter material. Heat-treated hardwood chips were used for bedding.
- Diet: Harlan TEKLAD certified Rodent 7012C, ad libitum
- Water: tap water, ad libitum
- Acclimation period: no less than 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 72 ± 3°F
- Humidity (%): 50 ± 20
- Air: air-conditioned
- Photoperiod (hrs dark/hrs light) : 12/12

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil

Details on exposure:

The test article-vehicle mixture, the vehicle alone, or the positive control was administered by a single intraperitoneal injection at a dose volume of 20 mL/kg body weight.

Duration of treatment / exposure:

Single injection

Frequency of treatment:

Single injection

Post exposure period:

24 or 48 hours

Remarks:

Doses / Concentrations:
125, 250, 500 mg/kg
Basis:
other: concentration injected

No. of animals per sex per dose:

- Vehicle: 10
- Low dose (125 mg/kg): 5
- Mid dose (250 mg/kg): 5
- High dose (500 mg/kg): 10
- Positive control: 5

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: intraperitoneal
- Doses / concentrations: 2.5 mg/mL

Tissues and cell types examined:

- All mice were observed after dose administration for clinical signs of toxicity.
- Bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Based upon the results of the toxicity study, the high dose for the micronucleus test was set at 500 mg/kg, which estimated to be the maximum tolerated dose.

TREATMENT AND SAMPLING TIMES
Bone marrow was collected 24h after dosing in all animals and also after 48 hours in the vehicle and high dose group. At the scheduled sacrifice times, five mice per sex per treatment were sacrificed by CO2 asphyxiation. Immediately following sacrifice, the femurs were distally exposed, cut just above the knee, and the bone marrow cells were transferred to a capped centrifuge tube containing approximately 1 mL fetal bovine serum.

DETAILS OF SLIDE PREPARATION:
The bone marrow cells were pelleted by centrifugation at approximately 100 x g for five minutes and the supernatant was drawn off, leaving a small amount of serum with the remaining cell pellet. The cells were resuspended by aspiration with a capillary pipet and a small drop of bone marrow suspension was spread onto a clean glass slide. Two slides were prepared from each mouse. The slides were fixed in methanol, stained with May-Gruenwald-Giemsa and permanently mounted.

METHOD OF ANALYSIS:
Bone marrow cells, polychromatic erythrocytes (PCEs) and normochromatic erythrocytes (NCEs) were analyzed for the presence of micronuclei. Using medium magnification (10 x 40), an area of acceptable quality was selected such that the cells were well spread and stained (10 x 100). 2000 PCEs per animal were scored for the presence of micronuclei. The number of micronucleated NCEs in the field of 2000 PCEs was enumerated for each animal in order to assess the quality of the differential staining procedure. The proportion of PCEs to total erythrocytes was also recorded to quantify the proliferation state of the bone marrow.

Evaluation criteria:

Validity critertia: The mean incidence of micronuclei containing PCEs must not exceed 5/1000 PCEs (0.5%) in the vehicle control. The incidence of micronuclei containing PCEs in the positive control group must be significantly increased relative to the vehicle group (p≤0.05).
Evaluation criteria: The test article was considered to induce a positive response if a dose-responsive increase in micronuclei containing PCEs was observed and one or more doses were statistically elevated relative to the vehicle control. However, values that were statistically significant but did not exceed the range of historical negative or vehicle controls were judged as not biologically significant.

Statistics:

Statistical significance was determined using the Kastenbaum-Bowman tables which are based on the binomial distribution. All analyses were performed separately for each sex and sampling time.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Pilot study: All mice at 2000 mg/kg were found dead on the day of dosing. Clinical signs, consisting of piloerection, were observed in males at 1, 10, 100 and 1000 mg/kg. In addition, loss in body weight of approximately 10% was observed in male mice at 1000 mg/kg. Due to mortality at 2000 mg/kg, a toxicity study was performed.
- Toxicity study: Clinical signs following dose administration included: piloerection, prostration, irregular breathing in males and females at 500 and 1000 mg/kg and lethargy in males at 1000 mg/kg. Loss in body weight from approximately 2% to 14% was observed in male and female mice at 500 and 1000 mg/kg. Based upon these results, the high dose for the micronucleus test was set at 500 mg/kg, which was estimated to be the maximum tolerated dose.

RESULTS OF DEFINITIVE STUDY
- Mortality and clinical signs: No mortality was observed in all exposed animals. Clinical signs following dose administration included piloerection in male and female mice at all doses and lethargy in males and females at 250 mg/kg. In addition, prostration and irregular breathing were observed in males and females at 500 mg/kg.
- Induction of micronuclei: No appreciable reductions in the ratio of PCEs to total erythrocytes was observed in the test article-treated groups relative to the vehicle control groups suggesting that the test article did not inhibit erythropoiesis. The number of micronucleated PCEs per 10000 PCEs in the test article-treated groups was not statistically increased relative to the respective vehicle controls in either male or female mice, regardless of dose level or bone marrow collection time.
- Ratio of PCE/NCE: No appreciable increase in the number of micronucleated NCEs in the field of 2000 PCEs per animal was found indication that an optimal differential staining was achieved.

RESULTS OF CONTROLS
Cyclophosphamide induced a significant increase in micronucleated PCEs in both male and female mice. The negative and positive controls were consistent with the historical control data, indicating that there was no problem with the test system or quality of the test.

Conclusions:

Interpretation of results (migrated information): negative
Under the conditions of this test, the test substance was concluded to be negative in the mouse micronucleus assay using ICR mice.

Executive summary:

In a GLP compliant mouse micronucleus assay according to OECD 474, ICR mice were exposed to the test substance by intraperitoneal injection. Five mice per sex were treated with 125, 250 or 500 mg/kg test substance, vehicle or positive control article and sacrificed after 24 hours. iIn addition, 5 mice per sex treated with the vehicle or 500 mg/kg test substance were sacrificed after 48 hours. No mortality was observed in any male or female mice. Clinical signs following dose administration included piloerection in male and female mice at all doses and lethargy in males and females at 250 mg/kg. In addition, prostration and irregular breathing were observed in males and females at 500 mg/kg bw. No appreciable reductions in the ratio of polychromatic erythrocytes to total erythrocytes was observed in the test article-treated groups relative to the vehicle groups suggesting that the test substance did not inhibit erythropoiesis. No significant increase in micronucleated polychromatic erythrocytes in test article-treated groups relative to the respective vehicle control groups was observed in male or female mice at 24 or 28 hours after dose administration. The positive control induced a significant increase in micronucleated polychromatic erythrocytes in both male and female mice. Therefore, the test substance was concluded to be negative in the mouse micronucleus assay using ICR mice.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In a GLP compliant OECD 471 study the mutagenic potential of the test substance in the Bacterial Reverse Mutation Test using five strains of Salmonella typhimurium (TA98, TA100, TA102, TA1535 and TA1537) was performed (Himmelsbach, 2022). The test was performed in three valid experiments in the presence and absence of metabolic activation, with +S9 standing for the presence of a metabolic activation, and -S9 standing for absence of metabolic activation. In the first experiment, the test item (dissolved in Dimethyl sulfoxide, DMSO) was tested up to concentrations of 5000 µg/plate in the absence and presence of S9 mix in the strains TA98, TA100, TA102, TA1535 and TA1537 using the plate incorporation method. The test item showed no precipitates on the plates at any of the concentrations. Cytotoxicity could be observed in the presence and the absence of metabolic activation in all tested strains. The results of this experiment showed that none of the tested concentrations induced a relevant or concentration-related increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation. Based on the toxicity results of the Experiment 1, the experiment was repeated under the same conditions with adapted concentrations for the strains TA98 (-/+S9), TA100 (-/+S9), TA102 (-S9), TA1535 (-S9) and TA1537 (-/+S9). The test item (dissolved in DMSO) was tested up to concentrations of 1500 µg/plate in the presence and absence of metabolic activation. The test item showed no precipitates on the plates at any of the concentrations. Cytotoxicity could be observed in the presence and the absence of metabolic activation in all tested strains. The results of this experiment showed that none of the tested concentrations induced a relevant or concentration-related increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation. Based on the results of the Exp. 1 and 1b, the test item was tested up to concentrations of 5000 µg/plate in the presence and absence of S9 mix in all bacteria strains using the preincubation method. The test item showed no precipitates on the plates at any of the concentrations. Cytotoxicity could be observed in the presence and the absence of metabolic activation in all tested strains. The results of this experiment showed that none of the tested concentrations induced a relevant or concentration-related increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation. Based on the results of this study it is concluded that the test substance is not mutagenic in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 in the presence and absence of metabolic activation under the experimental conditions in this study.

In a GLP compliant in vitro Mouse Lymphoma Assay, L5178Y cells were exposed to the test substance with and without metabolic activation (S9 mix) to investigate the potential of the test substance to induce gene mutations (Bioreliance, 2002). The cells were treated for 4 and 24 hours in the non-activated test system, and for 4 h in the S9 activated test system. Different dosages (up to 100 and 150 µg/mL, with and without metabolic activation, respectively) were chosen. Cytotoxicity (total growth ≤50% of the solvent control) was observed at ≥75 and 150 µg/mL without activation with 4 h and 24 h exposure, respectively, and at dose levels ≥30 µg/mL with metabolic activation. In the presence of S9 mix the test substance induced in one culture (40 µg/mL) a mutant frequency between 55 and 99 over solvent control. All other mutant frequencies were below 55 over solvent control and therefore the results were negative in the presence of S9 -mix. In the absence of S9 mix results were equivocal, because in both the 4 h and 24 h treatment one cultures exhibited a mutant frequency of ≥100 mutants per 1E6 clonable cells over that of the solvent control. However, no dose-response was observed, but as for both exposure times several cultures exhibited mutant frequencies between 55 and 99 mutants per 1E6 clonable cells over that of the solvent control, the results in the absence of a metabolic activation system were considered to be equivocal.

In a GLP-compliant chromosome aberration test Chinese hamster ovary (CHO) cells were exposed to the test substance in DMSO with and without metabolic activation (S9 -mix) (Bioreliance, 2002). The cells were treated for 4 and 20 h in the non-activated test system and for 4 h in the S9 activated test system and all cells were harvested at 20 h after treatment initiation. Different dosages (up to 200, 300 and 400 µg/mL, for +S9 mix 4 h, -S9 mix 4 h and -S9 mix 20 h, respectively) were chosen in the three groups. Cytotoxicity was observed in all three groups at the highest tested dose. The percentage of cells with structural aberrations in the test article-treated groups was significantly increased above that of the solvent control at dose levels 100 and 200 µg/mL in the S9 activated 4 hour exposure group. The precentage of numerical aberrations was not significantly increased. In the non-activated 4 h exposure group, the percentage of cells with structural aberrations was significantly above that of the solvent control at dose level 300 µg/mL. However, this percentage of 3.0 % was within the historical solvent control and therefore not considered to be biologically relevant. In the absence of the S9 mix in the 20 h exposure group, the test article did not increase the frequency of cells with aberrations. Based on these results the test substance is considered to be mutagenic in the presence of metabolic activation.

In a GLP compliant mouse micronucleus assay according to OECD 474, ICR mice were exposed to the test substance by intraperitoneal injection (Bioreliance, 2002). Five mice per sex were treated with 125, 250 or 500 mg/kg bw test substance, vehicle or positive control article and sacrificed after 24 h. iIn addition, 5 mice per sex treated with the vehicle or 500 mg/kg bw test substance were sacrificed after 48 h. No mortality was observed in any male or female mice. Clinical signs following dose administration included piloerection in male and female mice at all doses and lethargy in males and females at 250 mg/kg bw. In addition, prostration and irregular breathing were observed in males and females at 500 mg/kg bw. No appreciable reductions in the ratio of polychromatic erythrocytes to total erythrocytes was observed in the test article-treated groups relative to the vehicle groups suggesting that the test substance did not inhibit erythropoiesis. No significant increase in micronucleated polychromatic erythrocytes in test article-treated groups relative to the respective vehicle control groups was observed in male or female mice at 24 or 28 h after dose administration. The positive control induced a significant increase in micronucleated polychromatic erythrocytes in both male and female mice. Therefore, the test substance was concluded to be negative in the mouse micronucleus assay using ICR mice.

 

Justification for classification or non-classification

The compositions of the Substance named “Mesitol technical grade” contain an impurity of phenol (EC 203-632-7, CAS: 108-95-2) with the concentration range > 0% - ≤ 3% w/w. Phenol is included in Annex VI of Regulation (EC) No 1272/2008 (CLP) as mutagen, Category 2 (Muta 2) (H341) with a statement “Suspected of causing genetic defects” (harmonized classification).

Since the upper value of the concentration range is higher than the limit of 1% for mutagen, Category 2 (Table 3.5.2. of Annex I to CLP), the Mesitol technical grade should therefore also be classified as mutagen, Category 2 (H341) according to Article 10(1) and to section 3.5.3.1.1. of Annex I to CLP.