4-hydroxy-4-methylpentan-2-one

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The genetic toxicity of diacetone alcohol (DAA) has been assessed in 7 in vitro studies, including 3 bacterial reverse mutation assays, 2 mammalian gene mutation assay, and 2 mammalian chromosome aberration test. Negative results were reported in all studies.

Bacterial gene mutation assay

In the key bacterial reverse mutation assay (according to OECD guideline 471), DAA was tested in a study at doses of 0, 313, 625, 1,250, 2,500, or 5,000 µg/plate in Salmonella typhimurium strains TA 98, TA 100, TA 1535, and TA 1537 and Escherichia coli strain WP2 uvr A both in the absence and presence of exogenous metabolic activation (phenobarbital and 5,6-benzoflavone-induced rat liver S9) (MHW, 1997).  Incubations at each concentration were done in triplicate and an independent repeat experiment was performed. Japanese Pharmacopeia water for injection was used as the vehicle and positive controls were included in all incubations.  No cytotoxicity and no increase in the reverse mutation rate were observed at any DAA concentration in any of the tester strains either in the absence or presence of metabolic activation.  Incubation with positive control substances either in the absence or presence of metabolic activation resulted in anticipated increases in the reverse mutation rate.

In a supportive non-GLP bacterial reverse mutation assay (equivalent to OECD guideline 471), DAA was tested at doses of 0, 31.25, 62.5, 125, 250, 500, 1,000, 2,000, or 4,000 µg/plate in S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537, and TA 1538 and Escherichia coli strains WP2 and WP2 uvr A both in the absence and presence of exogenous metabolic activation (Aroclor 1254-induced rat liver S9) (Priston et al., 1983).  The experiment was conducted in triplicate and an independent repeat experiment was performed. Sterile distilled water was used as the vehicle and positive controls were included in all incubations. No cytotoxicity was observed and no increase in the reverse mutation rate was noted at any DAA concentration either in the absence or presence of metabolic activation. Incubation with positive control substances in the absence or presence of metabolic activation did not always result in anticipated increases in reverse mutation rates and, for this reason, the study is considered to be reliable with restrictions.

In another supportive bacterial reverse mutation assay (equivalent to OECD guideline 471), DAA was tested at doses of 0, 100, 333, 1,000, 3,333, or 10,000 µg/plate in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537, and TA 1538 both in the absence and presence of exogenous metabolic activation (Aroclor 1254-induced rat liver S9) (Seifried et al., 2006).  Incubations at each concentration were done in triplicate; however, an independent repeat experiment was not performed. Water was used as the vehicle (it was not reported if water was distilled, nanopure, or tap) and positive controls were included in all incubations (specific compounds were not reported).  No cytotoxicity and no increase in the reverse mutation rate were observed at any DAA concentration in any of the tester strains either in the absence or presence of metabolic activation.  Incubation with positive control substances either in the absence or presence of metabolic activation resulted in anticipated increases in the reverse mutation rate.  This study is considered to be reliable with restrictions due to the missing S. typhimuriumstrain TA 102 orE. coli strain WP2 uvr A.

Chromosomal aberration assay

In a key mammalian chromosome aberration test (according to OECD guideline 473), DAA was tested at doses of 0, 0.3, 0.6, and 1.2 mg/mL both in the absence and presence of exogenous metabolic activation (phenobarbital and 5,6-benzoflavone-induced rat liver microsomes) in CHL/IU cells (MHW, 1997).  Incubations at each concentration were done in quadruplicate; however, an independent repeat experiment was not performed.  Distilled injection water was used as the vehicle and mitomycin C and cylophosphamide were used as the positive control compounds in the absence and presence of metabolic activation, respectively.  No cytotoxicity was observed and no chromosome damage was noted at any DAA concentration.  Incubations with the positive control compounds resulted in anticipated increases in chromatid damage.

In an in vitro chromosomal aberration assay performed with rat liver (RL4) cells, diacetone alcohol induced a slight increase in chromatid damage, breaks and fragments but no dose-response relationship was established (Brook et al., 1988). However, the result was judged negative by the AFC expert panel (The EFSA Journal (2004) 166, 1-44).

Mammalian cell gene mutation assay

In a key study, the potential of Diacetone alcohol to induce mutations at the TK (Thymidine Kinase) locus in L5178Y mouse lymphoma cells was evaluated according to the international guidelines and in compliance with GLP (Sire, 2010). After a preliminary toxicity test, Diacetone alcohol was tested at concentrations of 0, 156.3, 312.5, 625, 1250, 2500 and 5000 µg/mL, in two independent experiments, with and without a metabolic activation system prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. Without S9 mix, no noteworthy decrease in Adj. RTG and increase in the mutation frequency were noted at any tested dose-levels either following the 3- or the 24-hour treatment. With S9 mix, a 29-44% decrease in the Adj. RTG. was noted in the first experiment, but none in the second experiment and no noteworthy increase in the mutation frequency was induced at any of the tested dose-levels, in either experiment. Diacetone alcohol did not show any mutagenic activity in the mouse lymphoma assay.

In a supportive mammalian gene mutation assay (equivalent to OECD guideline 476), DAA was tested at doses of 0, 3,000, 3,500, 4,000, 4,500, or 5,000 µg/mL both in the absence and presence of exogenous metabolic activation (Aroclor 1254-induced rat liver S9) in mouse lymphoma L5178Y cells (Seifried et al., 2006). The experiment was conducted in duplicate; however, an independent repeat experiment was not performed. Water was used as the vehicle (it was not reported if the water was distilled, nanopure, or tap) and positive control compounds were included in all incubations (specific compounds were not reported). No cytotoxicity and no increase in the mutant frequency were observed at any DAA concentration either in the absence or presence of metabolic activation. Incubation with positive control substances in the absence or presence of metabolic activation were reported to result in anticipated increases in the mutation frequencies.

Link to relevant study records

Reference

Reference 1

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:

1997

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:

1981

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Deviations:

no

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine opero

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital and 5,6-benzoflavone-induced rat liver microsomes (S9)

Test concentrations with justification for top dose:

0, 313, 625, 1250, 2500, or 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Japanese Pharmacopeia water for injection

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Japanese Pharmacopeia water for injection

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: - See Table 1

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 minutes at 37°C
- Exposure duration: 48 hours at 37°C

NUMBER OF REPLICATIONS: 3 plates for both the solvent and the positive control groups in the dose range finding tests and 1 for each dose. Additionally, during this study, 3 plates were used for each of the control groups and each dose. Independent repeat performed.
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

DETERMINATION OF CYTOTOXICITY
Dose levels: 0, 50, 150, 500, 1500, or 5000 µg/plate (±S9)

Evaluation criteria:

Of the five types of assay strains, under the conditions with and without the S9 mix in at least one type of assay strain, if the mean values for the number of mutant colonies on a plate containing the test substance were more than double than those of the solvent control when verifying the reproducibility and dose dependence, the test substance was deemed to be mutagenic (positive) for this study. If only one of these two tests confirmed a dose at which the mean number of colonies was double or more that of the solvent control values, and if the solvent control value was 10 or less, dose dependency could not be confirmed by an increase in the number of mutant colonies, it was deemed to be negative.

Statistics:

None required (number of revertant colonies counted).

Species / strain:

S. typhimurium TA 100

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 applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

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 applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

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 applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

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 applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

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 applicable

Positive controls validity:

valid

Conclusions:

Interpretation of results : negative

Executive summary:

DAA was tested a bacterial reverse mutation assay (according to OECD guideline 471) at doses of 0, 313, 625, 1,250, 2,500, or 5,000 µg/plate in Salmonella typhimurium strains TA 98, TA 100, TA 1535, and TA 1537 and Escherichia coli strain WP2 uvr A both in the absence and presence of exogenous metabolic activation (phenobarbital and 5,6-benzoflavone-induced rat liver S9).  Incubations at each concentration were done in triplicate and an independent repeat experiment was performed. Water for injection was used as the vehicle and positive controls were included in all incubations. No cytotoxicity and no increase in the reverse mutation rate were observed at any DAA concentration in any of the tester strains either in the absence or presence of metabolic activation. Incubation with positive control substances either in the absence or presence of metabolic activation resulted in anticipated increases in the reverse mutation rate. 

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented, according to accepted guidelines

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1987

Deviations:

no

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

primary culture, other: CHL/IU cells

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital and 5,6-benzoflavone-induced rat liver microsomes (S9)

Test concentrations with justification for top dose:

Continuous treatment (24 hours; -S9): 0, 0.3, 0.6, and 1.2 mg/mL
Continuous treatment (48 hours; -S9): 0, 0.3, 0.6, and 1.2 mg/mL
Short-term treatment (6 hours; ±S9): 0, 0.3, 0.6, and 1.2 mg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: distilled injection water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

- Distilled injection water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Cylophosphamide (+S9) and mitomycin C (-S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 6, 24, and 48 hours (-S9) and 6 hours (+S9)

SPINDLE INHIBITOR (cytogenetic assays): Colcemid

NUMBER OF REPLICATIONS: 4 dishes/concentration; no independent repeat performed

NUMBER OF CELLS EVALUATED: 200 cells/concentration

Evaluation criteria:

The chromosomal analysis was conducted based on the classification methods of The Mammalian Mutagenesis Study Group of the Environmental Mutagen Society of Japan (MMC). Broader and mid-division images were observed to prevent chromosomes from being lost. For each group, the number of cells observed, the type of chromosome and the number and type of chromosomal aberrations, and the number of ploidal cells were entered on the record form. Additionally, cells with aberrations were recorded on the record form indicating the position on the slide according to the microscopic stage location. Four sample slides were taken from one dish, and four observers analyzed them without knowing the treatment conditions. 200 of the cells with structural aberrations in a single group were analyzed mid-division, with 800 per group for ploidal cells.

Statistics:

Fisher’s direct probability test was conducted between the background data for the solvent and the test substance treatment group and significant differences examined with the familywise significant standard of 5%. If there is a significant difference using the direct probability method, the presence of dose dependency was verified using a Cochran Armitage trend test2) (p<0.05). The final judgment was performed based on statistical and biological evaluations.

Species / strain:

primary culture, other: CHL/IU cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

10 mM

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

Interpretation of results: negative

Executive summary:

In a mammalian chromosome aberration test (according to OECD guideline 473), DAA was tested at doses of 0, 0.3, 0.6, and 1.2 mg/mL both in the absence and presence of exogenous metabolic activation (phenobarbital and 5,6-benzoflavone-induced rat liver microsomes) in CHL/IU cells.  Incubations at each concentration were done in quadruplicate; however, an independent repeat experiment was not performed.  Distilled injection water was used as the vehicle and mitomycin C and cylophosphamide were used as the positive control compounds in the absence and presence of metabolic activation, respectively.  No cytotoxicity was observed and no chromosome damage was noted at any DAA concentration.  Incubations with the positive control compounds resulted in anticipated increases in chromatid damage.

Reference 3

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

Study period:

23 February 2009 - 15 June 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Compliant to GLP and testing guideline; adequate coherence between data, comments and conclusions.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1997

Deviations:

yes

Remarks:

For the preliminary test and the first experiment of the study, the dosage forms were kept at room temperature instead of at +4°C, during the 15 minutes between their preparation and their use in the treatment.

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

For the preliminary test and the first experiment of the study, the dosage forms were kept at room temperature instead of at +4°C, during the 15 minutes between their preparation and their use in the treatment.

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 (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and sodium
pyruvate (200 µg/mL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: 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:

156.3, 312.5, 625, 1250, 2500 and 5000 µg/mL.

Vehicle / solvent:

Vehicle used: RPMI culture medium

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

RPMI

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without S9 mix: Methylmethane Sulfonate (MMS). With S9 mix: Cyclophosphamide (CPA)

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): trifluorothimidine

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth.

Evaluation criteria:

IWGT recommendations (d) 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%, is not considered as positive result.
A test item is determined to be non-mutagenic when there is no culture showing an Adj. RTG value between 10-20% if (e):
- 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:

no 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: 500 mg/mL
- Precipitation: no precipitate at the final dose-level of 5000 µg/mL;pH was approximately 7.6 (as for the vehicle control) and the osmolality equal
to 355 mOsm/kg H2O (306 for the vehicle control).

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 below for the mutagenicity experiment.
The test item was freely soluble in the vehicle (RPMI) at 500 mg/mL.

Using a treatment volume of 200 µL/20 mL of culture medium, the final dose-level of 5000 µg/mL showed no precipitate. At this dose-level, the pH was approximately 7.6 (as for the vehicle control) and the osmolality equal to 355 mOsm/kg H2O (306 for the vehicle control).

The dose-levels used for treatment were 10, 100, 500, 1000, 2500, 5000 µg/mL.

Following the 3-hour treatment without S9 mix, a 29-50% decrease in adjusted relative survival (Adj. RTG) was noted at all dose-levels.
Following the 24-hour treatment without S9 mix, no noteworthy decrease in Adj. RTG. was noted at any tested dose-levels.
Following the 3-hour treatment with S9 mix, no noteworthy decrease in Adj. RTG. was noted at any tested dose-levels.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Experiments without S9 mix:
Using a treatment volume of 200 µL/20 mL, the selected dose-levels were 156.3, 312.5, 625, 1250, 2500 and 5000 µg/mL for the first experiment (3-hour treatment) and the second experiment (24 hour treatment).

Cytotoxicity:
No noteworthy decrease in Adj. RTG. was noted at any tested dose-levels either following the 3 or the 24-hour treatment.

Mutagenicity:
No noteworthy increase in the mutation frequency was induced at any of the tested dose-levels, either following the 3-hour treatment or the
following the 24-hour treatment.

Experiments with S9 mix:
Using a treatment volume of 200 µL/20 mL, the selected dose-levels were 156.3, 312.5, 625, 1250, 2500 and 5000 µg/mL for both mutagenicity
experiments.

Cytotoxicity:
In the first experiment, a 29-44% decrease in the Adj. RTG. was noted without any clear evidence of a dose-response relationship.
In the second experiment, no noteworthy decrease in the Adj. RTG. was induced at any dose levels.

Mutagenicity:
No noteworthy increase in the mutation frequency was induced at any of the tested dose-levels, in either experiment.

Conclusions:

The test item Diacetone alcohol did not show any mutagenic activity in the mouse lymphoma assay.

Executive summary:

The potential of Diacetone alcohol to induce mutations at the TK (Thymidine Kinase) locus in L5178Y mouse lymphoma cells was evaluated according to the international guidelines and in compliance with GLP. After a preliminary toxicity test, Diacetone alcohol was tested at concentrations of 0, 156.3, 312.5, 625, 1250, 2500 and 5000 µg/mL, in two independent experiments, with and without a metabolic activation system prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. Without S9 mix, no noteworthy decrease in Adj. RTG and increase in the mutation frequency were noted at any tested dose-levels either following the 3- or the 24-hour treatment. With S9 mix, a 29-44% decrease in theAdj. RTG. was noted in the first experiment, but none in the second experiment and no noteworthy increase in the mutation frequency was induced at any of the tested dose-levels, in either experiment. Diacetone alcohol did not show any mutagenic activity in the mouse lymphoma assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The substance does not meet the criteria for classification and labeling for this endpoint, as set out in Regulation (EC) NO. 1272/2008.