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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

MESITYL OXIDE did not show any mutagenic activity in the mouse lymphoma assay (Sire, 2009 -OECD 476) and in an ames test (OECD 471, Bisini, 2010)

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:
June 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: fully GLP compliance
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
S. typhimurium tester strains: histidine operon - E. coli: tryptophan operon
Species / strain / cell type:
other: Salmonella typhimurium TA1535, TA1537, TA98, TA100 and Escherichia coli WP2 uvrA
Additional strain / cell type characteristics:
other: uvrA, uvrB: Sensitivity to UV irradiation. rfa: Sensitivity to Crystal Violet. pKM101: Resistance to Ampicillin.
Metabolic activation:
with and without
Metabolic activation system:
S9 rat liver tissue fraction - Inducing Agents :Phenobarbital – 5,6-Benzoflavone
Test concentrations with justification for top dose:
Preliminary toxicity test: 5000, 2500, 500, 158 and 50.0 µg/ml
Main Assay I (plate incorporation): 5000, 2500, 1250, 625 and 313 µg/plate
Main Assay II (pre-incubation): 5000, 2500, 1250, 625 and 313 µg/plate
Vehicle / solvent:
dimethylsulfoxide (DMSO)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Sodium azide: TA1535, TA100 without S9 metabolism; 9-amino-acridine TA1537 without S9 metabolism; methylmethanesulphonate WP2 uvrA without S9 metabolism; 2-aminoanthracene all tester strains with S9 metabolism
Details on test system and experimental conditions:
METHOD OF APPLICATION: preliminary toxicity test and main assay I in agar plate incorporation; main assay II preincubation

DURATION
- Preincubation period: 30 minutes at 37 °C
- Incubation time: approximately 72 hours at 37 °C

NUMBER OF REPLICATIONS: three replicate plates for each experimental point

NUMBER OF CELLS EVALUATED: 100 - 500 million of viable bacteria/plate (titre) for each strain.

DETERMINATION OF CYTOTOXICITY
Reduction of revertant numbers and/or thinning of the background lawn

Evaluation criteria:
For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.
Statistics:
Regression lines, after square root transformation, are calculated using a minimum of the three lowest dose levels and then including the further dose levels in turn. The correlation co-efficient (r), the value of students "t" statistic and the p-value for the regression lines are also calculated.
Species / strain:
other: Salmonella typhimurium TA1535, TA1537, TA98, TA100 and Escherichia coli WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
Precipitation: No precipitation of the test item was observed at the end of the incubation period at any concentration.

RANGE-FINDING/SCREENING STUDIES:
No toxicity was observed up to 5000 µg/plate
COMPARISON WITH HISTORICAL CONTROL DATA: that mean plate counts for untreated and positive control plates fell within the RTC historical control range
ADDITIONAL INFORMATION ON CYTOTOXICITY:
Main Assay I (plate incorporation method): No toxicity was observed with any tester strain at any dose level, in the absence or presence of S9 metabolism.
Main II (pre-incubation method): Toxicity was observed with all tester strains at the two or three highest concentrations, in the absence and presence of S9 metabolism

No two-fold increases in the number of revertant colonies were observed after treatment at any dose level, in any tester strain, in the absence or presence of S9 metabolism. It must be concluded that the test item Mesityl oxide is not mutagenic to S. typhimurium or E. coli under the reported experimental conditions.

Conclusions:
Interpretation of results (migrated information):
negative

It is concluded that the test item Mesityl oxide does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.
Executive summary:

The test item Mesityl oxide was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli,as measured by reversion of auxotrophic strains to prototrophy. The five tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone.  The test item was used as a solution in dimethylsulfoxide (DMSO). Mesityl oxide was assayed in the toxicity test at a maximum concentration of 5000 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 µg/plate. No precipitation of the test item was observed at the end of the incubation period at any concentration. No toxicity was observed with any tester strain at any dose level in the absence or presence of S9 metabolism.   In Main Assay I, using the plate incorporation method, the test item was assayed at the maximum dose level of 5000 µg/plate and at four lower dose levels spaced by two-fold dilutions: 2500, 1250, 625 and 313 µg/plate. No toxicity was observed with any tester strain at any dose level, in the absence or presence of S9 metabolism. No precipitation of the test item was observed at the end of the incubation period at any concentration. As no increases in revertant numbers were observed at any concentration tested, a pre-incubation step was included for all treatments of Main Assay II. The test item was assayed at the same dose levels employed inToxicity was observed with all tester strains at the two or three highest concentrations, in the absence and presence of S9 metabolism. No precipitation of the test item was observed at the end of the incubation period at any concentration. The test item did not induce two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose level, in any tester strain, in the absence or presence of S9 metabolism. It is concluded that the test item Mesityl oxide does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 February 2008 to 25 November 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
no chemical analysis of the dosage forms was performed
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine Kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 medium containing L-Glutamine (2mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and sodium pyruvate (200 µg/mL).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Without S9 mix:
- first experiment (3-hour treatment): 0.313, 0.625, 1.25, 2.5, 5 and 10 mM
- second experiment (24-hour treatment): 0.078, 0.156, 0.313, 0.625, 1.25, 2.5 and 5 mM.
With S9 mix:
- first experiment: 0.313, 0.625, 1.25, 2.5, 5 and 10 mM
- second experiment: 1, 2, 4, 6, 8 and 10 mM.
Vehicle / solvent:
- Vehicle used: dimethylsulfoxide (DMSO).
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: methylmethane sulfonate (MMS) -S9; cyclophosphamide +S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 and 24 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 11-12 days

SELECTION AGENT (mutation assays): trifluorothymidine

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth.
Evaluation criteria:
IWGT recommendations were followed for the determination of a positive result which should fulfill the following criteria:
. at least at one dose-level the mutation frequency minus the mutation frequency of the vehicle control equals or exceeds the global evaluation factor (126 x 10-6 for the microtiter method),
. and a dose-related trend is demonstrated by a statistically significant trend test.

Unless considered as clearly positive, the reproducibility of a positive effect should be confirmed.
Noteworthy increases in the mutation frequency observed only at high levels of cytotoxicity (RTG lower than 10%), but with no evidence of mutagenicity at dose-levels with RTG between 10 and 20%, are considered as positive result.
A test item may be determined to be non-mutagenic when there is no culture showing an Adj. RTG value between 10-20% if:
. there is at least one negative data point between 20 and 25% Adj. RTG and no evidence on mutagenicity in a series of data points between 100 to 20% Adj. RTG,
. there is no evidence of mutagenicity in a series of data points between 100 to 25% and there is also a negative data point between 10 and 1% Adj. RTG
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
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
- Vehicle solubility: up to 196.28 mg/mL.
- Precipitation: no precipitate at the final dose-level of 981.4 µg/mL (which corresponds to 10 mM); pH of 7.3 and osmolality of 379 mOsm/kg H2O.

RANGE-FINDING/SCREENING STUDIES:
To assess the cytotoxicity of the test item, at least six dose-levels (one culture/dose-level) were tested both with and without metabolic activation.
A treatment of 3 hours (with and without S9 mix) and 24 hours (without S9 mix) was performed using a final concentration and conditions as described for the mutagenicity experiment.
Since the test item was freely soluble and non-severely toxic following the 3-hour treatment in the preliminary test with and without S9 mix at up to 10 mM, the highest dose-level selected for the main test using these experimental conditions was 10 mM, according to the criteria specified in the international guidelines.
Since the test item was toxic following the 24-hour treatment in the preliminary test with 0% Adj. RTG at 5 mM, the choice of the highest dose-level for the main test using these experimental conditions (5 mM) was based on the level of toxicity, according to the criteria specified in the international guidelines (decrease in Adj. RTG).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Experiments without S9 mix:
Following the 3-hour treatment, a slight to marked toxicity was induced at dose levels = 1.25 mM, as shown by 31 - 70% decrease in Adj. RTG.
Following the 24-hour treatment, a marked to severe toxicity was induced at dose levels = 2.5 mM, as shown by 70 - 100% decrease in Adj. RTG.
Experiments with S9 mix:
In the first experiment, a slight to marked toxicity was induced at dose levels = 0.625 mM as shown by 27 - 77% decrease in Adj. RTG.
In the second experiment, a slight to moderate toxicity was noted at dose-levels = 4 mM, as shown by 32 - 48% decrease in Adj. RTG.

MUTAGENICITY:
Experiments without S9 mix:
Following the 3- and 24-hour treatments, no noteworthy increase in the mutation frequency was observed.
Experiments with S9 mix:
In the first experiment, a slight increase in the mutation frequency was observed at the dose-level of 10 mM. This increase (125 x 10-6 added to the vehicle control mean value of 102 x 10-6), even close to the global evaluation factor of 126 x 10-6, was not reproduced in the second experiment at the same dose-level. Therefore, it was not considered as biologically relevant.
No noteworthy increase in the mutation frequency was observed at any of the other dose-levels.
Remarks on result:
other: strain/cell type: L5178Y
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

MESITYL OXIDE did not show any mutagenic activity in the mouse lymphoma assay.
Executive summary:

The potential of the test item MESITYL OXIDE (purity: 99.91%) to induce mutations at the TK (thymidine kinase) locus in L5178Y mouse lymphoma cells was evaluated according to the international guidelines (OECD 476 and Commission Directive No. B17) and in compliance with the Principles of Good Laboratory Practice.

After a preliminary toxicity test, MESITYL OXIDE was tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.

Approximately 0.5 x 106 (3-hour treatment) or 0.15 x 106 (24-hour treatment) cells/mL in 20 mL culture medium with 5% horse serum were exposed to the test or control items, in the presence or absence of S9 mix (final concentration of S9 fraction 2%), at 37°C. For the 24-hour treatment, the incubation at 37°C was performed with a gentle shaking.

Cytotoxicity was measured by assessement of adjusted relative total growth (Adj. RTG) and relative suspension growth (Adj. RSG) as well as cloning efficiency following the expression time (CE2).

The number of mutant clones (differentiating small and large colonies) were checked after the expression of the mutant phenotype.

The test item was dissolved in dimethylsulfoxide (DMSO).

The dose-levels for the positive controls were as follows:

. without S9 mix: methylmethane sulfonate (MMS), used at a final concentration of 25 µg/mL (3-hour treatment) or 5 µg/mL (24-hour treatment),

. with S9 mix: Cyclophosphamide (CPA), used at a final concentration of 3 µg/mL.

In the culture medium, the final dose-level of 10 mM (corresponding to 981.4 µg/mL) showed no precipitate. At this dose-level, the pH and the osmolality values were comparable to those of the vehicle control culture.

The cloning efficiencies CE2 and the mutation frequencies of the vehicle and positive controls were as specified in acceptance criteria. The study was therefore considered valid.

Since the test item was freely soluble and non severely toxic following the 3-hour treatment in the preliminary test with and without S9 mix, the highest dose-level selected for the main test using these experimental conditions was 10 mM, according to the criteria specified in the international guidelines.

Since the test item was toxic following the 24-hour treatment in the preliminary test, the choice of the highest dose-level for the main test using these experimental conditions was based on the level of toxicity, according to the criteria specified in the international guidelines (decrease in Adj. RTG).

Experiments without S9 mix:

Using a treatment volume of 100 µL/20 mL, the selected dose-levels were as follows:

. 0.313, 0.625, 1.25, 2.5, 5 and 10 mM for the first experiment (3-hour treatment),

. 0.078, 0.156, 0.313, 0.625, 1.25, 2.5 and 5 mM for the second experiment (24-hour treatment).

Cytotoxicity:

Following the 3-hour treatment, a slight to marked toxicity was induced at dose-levels > 1.25 mM, as shown by 31 - 70% decrease in Adj. RTG.

Following the 24-hour treatment, a marked to severe toxicity was induced at dose-levels > 2.5 mM, as shown by 70 - 100% decrease in Adj. RTG.

Mutagenicity:

Following the 3- and 24-hour treatments, no noteworthy increase in the mutation frequency was observed.

Experiments with S9 mix:

Using a treatment volume of 100 µL/20 mL, the selected dose-levels were as follows:

. 0.313, 0.625, 1.25, 2.5, 5 and 10 mM for the first experiment,

. 1, 2, 4, 6, 8 and 10 mM for the second experiment.

Cytotoxicity:

In the first experiment, a slight to marked toxicity was induced a dose-levels > 0.625 mM as shown by 27 - 77% decrease in Adj. RTG.

In the second experiment, a slight to moderate toxicity was noted at dose-levels > 4 mM, as shown by 32 - 48% decrease in Adj. RTG.

Mutagenicity:

In the first experiment, a slight increase in the mutation frequency was observed at the dose-level of 10 mM. This increase (125 x 10-6added to the vehicle control mean value of 102 x 10-6), even close to the global evaluation factor of 126 x 10-6, was not reproduced in the second experiment at the same dose-level. Therefore, it was not considered as biologically relevant.

No noteworthy increase in the mutation frequency was observed at any of the other dose-levels.

The test item MESITYL OXIDE did not show any mutagenic activity in the mouse lymphoma assay.

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

Genetic toxicity in vivo

Description of key information

Mesityl oxide is negative in the in vivo mouse bone marrow micronucleus assay at a dose level which produced deaths in the female animals and statistically significant bone marrow depression in the males (Barber, OECD 474, 1992)

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Inc., St. Constant, Canada
- Age at study initiation: , the males were approximately 65 days old and the females 56 days old at dosing
- Weight at study initiation: the weight range of the mice was 24.2 to 33.8 g.
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Fasting period before study: no
- Housing: two or three per cage
- Diet : Agway Prolab® RMII 3000 pellets ad libitum
- Water : tap water ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 72 ± 4
- Humidity (%): 50 ± 20
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To: 11/12/91 to 03/18/92
Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: solubility
- Concentration of test material in vehicle:
The test substance was analyzed using gas chromatography/mass spectrosoopy and the structure was confirmed as that of 4-methyl-3-pentene-2-one. The dosing solutions used in the micronucleus studies were analyzed by gas chromatography to confIrm their concentrations. The results are as follows:
Nominal Concentration Analytical Concentration Analytical/Nominal Percent
mg/g mg/ml
17.0 15.69 92.3
34.0 34.56 101.6
68.0 64.09 94.3
The mean ratio of the analytical concentration/nominal concentration was 0.928. Thus, the actual dose levels of mesityl oxide in these studies are lower by about 7 percent than the nominal dose levels given in the tables.
- Amount of vehicle (if gavage or dermal): no data
Duration of treatment / exposure:
Single administration
Post exposure period:
24, 48 and 72 hours
Remarks:
Doses / Concentrations:
170, 340 and 680 mg/kg bw
Basis:

No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide (40 mg/kg)
Tissues and cell types examined:
Polychromatic erythrocytes from the bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
The dose levels used in the micronucleus assay were chosen based upon the LD50 study. The initial study was begun on October 8, 1991. Based upon the reported ip LD50 for mesityl oxide of 354 mg/kg (Krasavage et al., 1982), groups of three males and three females were administered intraperitoneal doses of 500, 250 and 125 mg/kg mesityl oxide (in corn oil). Within 30 minutes after dosing, the males (3/3) receiving the high dose were ataxic and 1/3 high-dose, 1/3 low-dose and 2/3 mid-dose animals were lethargic. At 1.5 hours, none of the animals were ataxic, however, 1/3 of the low-dose and 2/3 mid-dose animals were lethargic. At 7 hours, all clinical signs were normal for the males and all survived the three-day observation period. For the females, those receiving the high dose (3/3) were ataxic at 15 minutes, and 2/3 of the mid-dose and 2/3 of the low-dose animals were lethargic. By 1.5 hours post-dose, 2/3 of the high-dose and one of the mid-dose animals were observed to be lethargic but none were ataxic. At 7 hours, all the females appeared normal and all survived the three-day observation period except one which died as the result of an accident.
A second study was begun on October 9, 1991 in which three males and three females were dosed at 750 mg/kg. All males and females were ataxic and lethargic at approximately 30 minutes after dosing, and one female died during this interval. By seven hours, all survivors were normal except one female that exhibited unkempt hair coat. Some ocular discharge was seen on the second day.
On October 21, 1991, groups of three males and three females were dosed with 1,000 and 875 mg/kg mesityl oxide. All six animals in the groups dosed with 1,000 mg/kg were found dead within 40 minutes. Two of the three males and one of the three females dosed at 875 mg/kg died during the first hour and another of the females was found dead at 48 hours. The remaining animals, 1 male and 1 female, survived the three-day observation period. The survival data were plotted on linear coordinates and the LD50 was estimated to be 850 mg/kg.
Therefore, the high dose in the micronucleus study was set at 680 mg/kg (0.8 times LD50), the mid-dose was set at one half of the high dose, or 340 mg/kg, and the low dose was one fourth of the high dose, or 170 mg/kg.

DETAILS OF SLIDE PREPARATION:
At the appropriate harvest time, the animals were euthanatized with CO2 and the adhering soft tissue and epiphyses of both femurs were removed. The marrow was flushed from the bone and transferred to centrifuge tubes containing 2 ml fetal calf serum (one tube for each animal). Following centrifugation to pellet the tissue, most of the supernatant was drawn off, the cells were resuspended, and the suspension spread on glass microscope slides and air-dried. The slides were then fixed in methanol, stained in Wright's-Giemsa stain, and rinsed in deionized water (Schmid, 1975). After being air-dried, the slides were coverslipped using mounting medium. Each slide was then re-labeled using ccded information generated by a computerized random number generation scheme. Thus, the identity of each slide was not known to those evaluating the slides.

Slides were scored for micronuclei and the relative numbers of polychromatic (PCE) and normochromatic (NCE) cells. One thousand PCEs per animal were routinely scored, except in cases of significant bone marrow depression. The frequency of micronucleated cells was expressed as number of micronucleated cells based on the total number of PCEs present in the microscopie fields. The frequency of PCEs versus NCEs was determined by counting the NCEs observed while scoring the 1,000 PCEs for micronuclei.
Evaluation criteria:
The criteria for determining a positive response involved a statistically significant dose-related increase in micronucleated PCEs, or the detection of a reproducible and statistically significant increase in micronucleated PCEs for at least one dose level. A test article that induced neither a statistically significant dose response nor a statistically significant and reproducible increase at one dose level was considered negative. In either case, the final decision was based on the scientific judgment of the Study Director.

Bone marrow depression was defined as a statistically significant decrease in the percent PCE ([PCE/PCE + NCE] x 100) in treated animals compared to the corresponding untreated control group.
Statistics:
The data were examined using plots, descriptive statistics, rank transformation, analysis of variance, Tukey's HSD Test and Dunnett's t-test. Statistical significance was detected using an alpha risk of <= 0.05.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
mortality and bone marrow depression at the top dose
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF DEFINITIVE STUDY
- Mortality and clinical signs
One female in the 24-hour harvest time group, two females in the 48-hour group and one female in the 72-hour group died within two hours of being dosed at 680 mg/kg. All other animals survived the treatments and provided bone marrow specimens for analysis at the appropriate harvest times. Hypothermia (1/5 females at 680 mg/kg), ocular discharges (all surviving mesityl oxide treated animals) and ptosis (all surviving mesityl oxide treated animals) were observed on the day of dosing_ Ocular discharges and ptosis were observed on the day following dosing for the 680 mg/kg animals. All animals appeared normal on subsequent examinations. Ataxia and lethargy were not observed during the micronucleus study.
- Induction of micronuclei (for Micronucleus assay):
The test article did not indure significant increases in micronucleated polychromatic erythrocytes (MNPCEs) in either the male or female mice, at the 24-, 48- or 72-hour harvest times when compared with the concurrent negative controls. Conversely, the positive control, CPA, induced highly significant increases in MNPCEs in both the male and female mite at 24 hours with concurrent bone marrow depression
- Ratio of PCE/NCE (for Micronucleus assay):
The test article produced statistically significant bone marrow dépression (decrease in the (PCE/NCE+PCE) ratio) at the 72-hour harvest time for the high-dose male mice.
Conclusions:
Interpretation of results : negative
Mesityl oxide is negative in the in vivo mouse bone marrow micronucleus assay at a dose level which produced deaths in the female animals and statistically significant bone marrow depression in the males.
Executive summary:

The ability of mesityl oxide to induce micronuclei in bone marrow polychromatic erythrocytes was evaluated after a single administration by intraperitoneal injection at 0, 170, 340, or 680 mg/kg to male and female Swiss CD-1 mice. The high dose of 680 mg/kg (80 % of the LD50) was chosen following a three-day study in which the LD50,was determined to be 850 mg/kg. The animals were dosed with the test substance and were sacrificed at 24, 48, and 72 hours after dosing for extraction of the bone marrow. Ten animals (five males and five females) were randomly assigned to each dose harvest time group. A negative control group (corn oil only) was included at each harvest time and a positive control group, dosed with cyclophosphamide (CPA) at 40 mg/kg, was included at the 24-hour sampling time only.

Mesityl oxide did not induce significant increases in micronucleated polychromatic erythrocytes (MNPCEs) in either the male or female mice, at the 24-, 48- or 72-hour harvest times when compared with the concurrent negative controls. Conversely, the positive control, CPA, induced highly significant increases in MNPCEs in both the male and female mice at 24 hours with concurrent bone marrow depression. In the micronucleus test, at the 680 mg/kg dose, one female in the 24-hour harvest time group, two females in the 48-hour harvest time group and one female in the 72-hour harvest time group died. The test article produced statistically significant bone marrow depression (decrease in the (PCE/NCE+PCE) ratio) at the 72-hour harvest time for the high-dose male mice. Therefore, it is concluded that mesityl oxide is negative in the in vivo mouse bone marrow micronucleus assay at a dose level which produced deaths in the female animals and statistically significant bone marrow depression in the males.

Additional information

Gene mutation assay

 

Mesityl oxide was examined for the ability to induce gene mutations in tester strains ofSalmonella typhimuriumandEscherichia coli,as measured by reversion of auxotrophic strains to prototrophy (Bisine, 2010). The five tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone.  The test item was used as a solution in dimethylsulfoxide (DMSO). Mesityl oxide was assayed in the toxicity test at a maximum concentration of 5000 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 µg/plate. No precipitation of the test item was observed at the end of the incubation period at any concentration. No toxicity was observed with any tester strain at any dose level in the absence or presence of S9 metabolism.   In Main Assay I, using the plate incorporation method, the test item was assayed at the maximum dose level of 5000 µg/plate and at four lower dose levels spaced by two-fold dilutions: 2500, 1250, 625 and 313 µg/plate. No toxicity was observed with any tester strain at any dose level, in the absence or presence of S9 metabolism. No precipitation of the test item was observed at the end of the incubation period at any concentration. As no increases in revertant numbers were observed at any concentration tested, a pre-incubation step was included for all treatments of Main Assay II. The test item was assayed at the same dose levels employed in Toxicity was observed with all tester strains at the two or three highest concentrations, in the absence and presence of S9 metabolism. No precipitation of the test item was observed at the end of the incubation period at any concentration. The test item did not induce two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose level, in any tester strain, in the absence or presence of S9 metabolism. It is concluded that Mesityl oxide does not induce reverse mutation inSalmonella typhimuriumorEscherichia coliin the absence or presence of S9 metabolism, under the reported experimental conditions.

 

The potential of mesityl oxide (purity: 99.91%) to induce mutations at the TK (thymidine kinase) locus in L5178Y mouse lymphoma cells was evaluated according to the international guidelines (OECD 476 and Commission Directive No. B17) and in compliance with the Principles of Good Laboratory Practice (Sire, 2009). After a preliminary toxicity test, mesityl oxide was tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. Approximately 0.5 x 106(3-hour treatment) or 0.15 x 106(24-hour treatment) cells/mL in 20 mL culture medium with 5% horse serum were exposed to the test or control items, in the presence or absence of S9 mix (final concentration of S9 fraction 2%), at 37°C. For the 24-hour treatment, the incubation at 37°C was performed with a gentle shaking. Cytotoxicity was measured by assessement of adjusted relative total growth (Adj. RTG) and relative suspension growth (Adj. RSG) as well as cloning efficiency following the expression time(CE2). The number of mutant clones (differentiating small and large colonies) were checked after the expression of the mutant phenotype. The test item was dissolved in dimethylsulfoxide (DMSO).

The dose-levels for the positive controls were as follows:

. without S9 mix: methylmethane sulfonate (MMS), used at a final concentration of 25 µg/mL (3-hour treatment) or 5 µg/mL (24-hour treatment),

. with S9 mix: Cyclophosphamide (CPA), used at a final concentration of 3 µg/mL.

In the culture medium, the final dose-level of 10 mM (corresponding to 981.4 µg/mL) showed no precipitate. At this dose-level, the pH and the osmolality values were comparable to those of the vehicle control culture. The cloning efficienciesCE2 and the mutation frequencies of the vehicle and positive controls were as specified in acceptance criteria. The study was therefore considered valid. Since the test item was freely soluble and non severely toxic following the 3-hour treatment in the preliminary test with and without S9 mix, the highest dose-level selected for the main test using these experimental conditions was 10 mM, according to the criteria specified in the international guidelines.

Since the test item was toxic following the 24-hour treatment in the preliminary test, the choice of the highest dose-level for the main test using these experimental conditions was based on the level of toxicity, according to the criteria specified in the international guidelines (decrease in Adj. RTG).

Without S9 mix, using a treatment volume of 100 µL/20 mL, the selected dose-levels were as follows:

. 0.313, 0.625, 1.25, 2.5, 5 and 10 mM for the first experiment (3-hour treatment),

. 0.078, 0.156, 0.313, 0.625, 1.25, 2.5 and 5 mM for the second experiment (24-hour treatment).

Following the 3-hour treatment, a slight to marked toxicity was induced at dose-levels>1.25 mM, as shown by 31 - 70% decrease in Adj. RTG. Following the 24-hour treatment, a marked to severe toxicity was induced at dose-levels>2.5 mM, as shown by 70 - 100% decrease in Adj. RTG.

Following the 3- and 24-hour treatments, no noteworthy increase in the mutation frequency was observed.

With S9 mix, using a treatment volume of 100 µL/20 mL, the selected dose-levels were as follows:

. 0.313, 0.625, 1.25, 2.5, 5 and 10 mM for the first experiment,

. 1, 2, 4, 6, 8 and 10 mM for the second experiment.

In the first experiment, a slight to marked toxicity was induced a dose-levels>0.625 mM as shown by 27 - 77% decrease in Adj. RTG. In the second experiment, a slight to moderate toxicity was noted at dose-levels>4 mM, as shown by 32 - 48% decrease in Adj. RTG.

In the first experiment, a slight increase in the mutation frequency was observed at the dose-level of 10 mM. This increase (125 x 10-6) added to the vehicle control mean value of 102 x 10-6, even close to the global evaluation factor of 126 x 10-6, was not reproduced in the second experiment at the same dose-level. Therefore, it was not considered as biologically relevant. No noteworthy increase in the mutation frequency was observed at any of the other dose-levels.

Mesityl oxide did not show any mutagenic activity in the mouse lymphoma assay.

 

Chromosomal aberration assay

 

A Micronucleus Test in the mouse was conducted according to OECD Guideline 474 (Barber, 1992). Mesityl oxide was administered by intraperitoneal injection at 0, 170, 340, or 680 mg/kg to groups of ten animals (five males and five females) that had been randomly assigned to each dose/harvest time group.  The high dose of 680 mg/kg (80 % of the LD50/3) was chosen following a three-day study in which the LD50/3 was determined to be 850 mg/kg. Mesityl oxide did not induce significant increases in micronucleated polychromatic erythrocytes (MNPCEs) in either the male or female mice, at the 24-, 48- or 72-hour harvest times when compared with the concurrent negative controls. At the 680 mg/kg dose, one female in the 24-hour harvest time group, two females in the 48-hour harvest time group and one female in the 72-hour harvest time group died. Mesityl oxide produced statistically significant bone marrow depression (decrease in the (PCE/NCE+PCE) ratio) at the 72-hour harvest time for the high-dose male mice. Therefore, it is concluded that mesityl oxide is negative in the in vivo mouse bone marrow micronucleus assay at a dose level which produced deaths in the female animals and statistically significant bone marrow depression in the males. 

 


Justification for selection of genetic toxicity endpoint
None selected, all tests negative.

Short description of key information:
Mesityl oxide has no genotoxic activity in a bacterial reverse mutation assay (OECD 471), in a mutation assay at the TK (thymidine kinase) locus in L5178Y mouse lymphoma cells (OECD 476) and in the micronucleus assay in bone marrow polychromatic erythrocytes of mouse (OECD 474).

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

No classification is warranted for germ cell mutagenicity according to Regulation no. 1272-2008 and Directive 67/548/EEC.