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Genetic toxicity in vitro

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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:
11.2009 - 04.2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Specific details on test material used for the study:
Test Material Name: Diisobutyl Ketone
Chemical Name; 2,6-Dimethyl-4-heptanone
Synonyms: DIBK
Supplier, City, State (Lot, Reference Number): The Dow Chemical Company, Freeport, Texas (Lot# XA2355T643)
Target gene:
Hypoxanthine-guanine-phosphoribosyltransferase (HPRT)
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CHO-K1-BH4

The cells are routinely maintained in Ham's F-12 nutrient mix supplemented with 5% (V/V) heat-inactivated (56°C, 30 minute), dialyzed fetal bovine serum, antibiotics, and antimycotics (penicillin G, 100 units/ml; streptomycin sulfate, 0.1 mg/ml; fungizone, 0.25 µg/ml), and an additional 2 mM L-glutamine. The selection medium used for the detection of HGPRT- mutants will be Ham's F-12 nutrient mix without hypoxanthine, supplemented with 10 µM 6-thioguanine and 5% serum and the above-mentioned antibiotics.
Metabolic activation:
with and without
Test concentrations with justification for top dose:
preliminary toxicity assay: 0 (solvent control), 5.7, 11.3, 22.7, 45.3, 90.6, 181.3, 362.5, 725, and 1450 μg/ml

mutagenicity assay: 0 (solvent control), 200, 400, 600, 800, 1000, 1200, and 1450 μg/ml in the absence of S9 and 0 (solvent control), 100, 200, 400, 500, 600, 700, 800, and 1000 μg/ml in the presence of S9

Vehicle / solvent:
The test material was first dissolved in DMSO and further diluted (1:100) with the treatment medium to obtain the desired concentrations.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 20-methylcholanthrene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 8 days
- Selection time (if incubation with a selection agent): 7 to 9 days

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency / colony formation
Evaluation criteria:
For an assay to be acceptable, the mutant frequency in positive controls should have been significantly higher than the solvent controls. An additional criteria was that the mutant frequency in the solvent controls should have been within reasonable limits of the laboratory historical control values and literature values. The test chemical was considered positive if it induced a statistically significant, dose related, reproducible increase in mutant frequency. The final interpretation of the data took into consideration such factors as the mutant frequency and cloning efficiencies in the solvent controls.
Statistics:
The frequency of mutants per 106 clonable cells was statistically evaluated using a weighted analysis of variance; weights were derived from the inverse of the mutant frequency variance. The actual plate counts are assumed to follow a Poisson distribution therefore the mean plate count was used as an estimate of variance. If the analysis of variance was significant at alpha = 0.05, a Dunnett's t-test was conducted, comparing each treated group and the positive control to the solvent control (alpha = 0.05, one-sided). Linear dose-related trend tests were performed
if any of the pairwise comparisons of test material with the solvent control yielded significant differences.
Species / strain:
Chinese hamster Ovary (CHO)
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:
The pH and osmolality of treatment medium containing approximately 2589 μg/ml of the test material and medium containing 1% DMSO were determined using a Denver Basic pH meter (Denver Instrument Co., Arvada, Colorado) and an OSMETTE A freezing point osmometer (Precision Systems, Inc., Natick, Massachusetts). Alterations in the pH and osmolality of the culture medium have been shown to induce false positive responses in in vitro genotoxicity assays. There was no appreciable change in the pH at this concentration as compared to the culture medium with solvent alone and the slight drop in the osmolality was interpreted to be inconsequential to the conduct of the assay (culture medium with the test material, pH = 7.38, osmolality = 435 mOsm/kgH2O; culture medium with 1% DMSO, pH = 7.39, osmolality = 463 mOsm/kgH2O).
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

The results of the CHO/HGPRT forward gene mutation assay with diisobutyl ketone indicated that under the conditions of this study, the test article was non-mutagenic when evaluated in the absence or presence of an externally supplied metabolic activation (S9) system.
Executive summary:

Diisobutyl ketone (2,6 dimethyl-4-heptanone) was evaluated in the in vitro Chinese hamster ovary cell/hypoxanthine-guanine-phosphoribosyl transferase (CHO/HGPRT) forward gene mutation assay. The genotoxic potential of the test material was assessed in two independent assays in the absence and presence of an externally supplied metabolic activation (S9) system. The concentrations ranged from 200 to 1450 μg/ml in the absence of S9 and from 100 to 1000 μg/ml in the presence of S9. The highest concentration for each activation system was based on the initial toxicity assay, where these concentrations resulted or exceeded relative cell survivals of 10-20%. The adequacy of the experimental conditions for detection of induced mutations was confirmed by employing positive control chemicals, ethyl methanesulfonate for assays in the absence of S9 and 20-methylcholanthrene for assays in the presence of S9. Solvent control cultures were treated with the solvent used to dissolve the test material (i.e. dimethyl sulfoxide). The results of the CHO/HGPRT forward gene mutation assay with diisobutyl ketone indicated that under the conditions of this study, the test article was non-mutagenic when evaluated in the absence or presence of an externally supplied metabolic activation (S9) system.

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:
1985
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): Di-lsobutyl ketone (DIBK)
- Physical state: Colourless, clear liquid
- Analytical purity: 2:1 mixture of 2,6-dimethyl-4-heptanone and 4,6-dimethyl-2-heptanone, water 0.2%
maximum
- Lot/batch No.: INDENT 9200/9943
Target gene:
Histidine operon for Salmonella typhimurium, Tryptophan operon for Escherichia coli
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
rat liver microsomal activation system (S9-mix)
Test concentrations with justification for top dose:
31.25, 62.5, 125, 250, 500, 1000, 2000 or 4000µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Solubility of test material
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA 1535 Migrated to IUCLID6: 2.5µL/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
TA 1538, TA 98 and TA 100 Migrated to IUCLID6: 10µg/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Potassium dichromate 10µg/plate
Remarks:
E.coli WP2 uvr A pKM 101
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Neutral red 10µg/plate
Remarks:
TA 1537
Details on test system and experimental conditions:
METHOD OF APPLICATION: Preincubation

DURATION
- Preincubation period: 30min
- Exposure duration: 48-72h


NUMBER OF REPLICATIONS: 2


DETERMINATION OF CYTOTOXICITY
- Method: other: colony count

Evaluation criteria:
No data
Statistics:
No data
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
At concentrations >500µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
At concentrations >500µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test compound, DIBK, produced no evidence of precipitation in the top agar up to 8000µg/ml showing that it was completely miscible in the aqueous test system at these concentrations.

RANGE-FINDING/SCREENING STUDIES: In a preliminary cytotoxicity assay with Salmonella typhimurium TA 100, DIBK was cytotoxic at concentrations above 500µg/ml in both the presence and in the absence of rat liver S9 fraction. Microscopical examination of the background lawn in the bacterial mutation assays showed some variation in cytotoxicity between the strains but, in general agreed with the observations using TA 100.


Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1: Maximum number of revertants for each strain compared to controls:

 

Maximum number of revertants (dose level [µg/mL])

solvent Control

positive control

DIBK treatment

Strain

With S9

Without S9

With S9

Without S9

With S9

Without S9

TA 98

30

14

331

20

31 (250)

15 (250)

TA 100

88

78

360

81

91 (62.5)

82 (31.25)

TA 1535

12

10

240

546

10 (125)

13 (500)

TA 1537

20

10

58

16

27 (250)

12 (62.5)

TA 1538

27

17

116

15

30 (250)

20 (62.5)

E. coli WP2

33

47

68

216

39 (62.5)

52 (125)

The addition of Diisobutyl Ketone (DIBK) at concentrations up to 4000 µg/ml to cultures of Escherichia coli. WP2 uvr A pkm 101, Salmonella typhimurium TA1535, TA1537, TA1538, TA98 or TA100 did not lead to any increase in the reverse gene mutation rate of these strains either in the presence or in the absence of rat liver S9 fraction under the conditions of this study.

Conclusions:
Interpretation of results (migrated information):
negative

The addition of Diisobutyl Ketone (DIBK) at concentrations up to 4000 µg/ml to cultures of Escherichia coli. WP2 uvr A pkm 101, Salmonella typhimurium TA1535, TA1537, TA1538, TA98 or TA100 did not lead to any increase in the reverse gene mutation rate of these strains either in the presence or in the absence of rat liver S9 fraction under the conditions of this study.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
only tested without metabolic activation
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
- Name of test material (as cited in study report): Di-lsobutyl ketone (DIBK)
- Physical state: Colourless, clear liquid
- Analytical purity: 2:1 mixture of 2,6-dimethyl-4-heptanone and 4,6-dimethyl-2-heptanone, water 0.2%
m/m maximum
- Lot/batch No.: INDENT 9200/9943
Target gene:
not applicable
Species / strain / cell type:
mammalian cell line, other: rat liver RL4 cells
Details on mammalian cell type (if applicable):
not available
Metabolic activation:
without
Test concentrations with justification for top dose:
62.5, 125, 250 and 500µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Solubility of test material in culture medium
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
without S9 Migrated to IUCLID6: 1µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium


NUMBER OF REPLICATIONS: 3


NUMBER OF CELLS EVALUATED: 300


DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
Evaluation criteria:
No data
Statistics:
No data
Species / strain:
mammalian cell line, other: Rat liver RL4 cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
At concentrations >400µg/mL as tested in range finding test
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
The only findings of note were two chromatid exchanges in one culture exposed to 125 µg/mL DIBK and a very small, apparently dose-related
increase in the frequency of chromatid gaps, but this is not considered to represent a compound-related effect, as the frequency of gaps was low
throughout. These observations are consistent with the amount of damage normally recorded in control cultures. The recorded incidence of polyploidy was high and variable in cultures. Although DIBK did not appear to affect this end point, it is considered that the changes would need to be large under these circumstances, and, therefore, the conclusions drawn from these results relate to structural chromosome aberrations only.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1: Metaphase chromosome analysis:

Conc. µg/mL

% Cells showing

Polyploidy

Chromatid gaps

Multiple aberrations

Severe damage

Chromatid aberrations

Chromosome aberrations

0

5.7

0.3

0.3

0

0.3

0

125

7.7

0.3

0

0

0.7

0

250

8.0

0.7

0

0

0

0

500

4.3

1.3

0

0

0.3

0

pos. control

4.5

13.5

2.0

2.5

10.0

0

The results show that Diisobutyl Ketone (DIBK) did not induce chromosome damage in rat liver(RL4)cells under the experimental conditions described. In the same experiment the positive control 7,12-dimethylbenzanthracene (DMBA) induced chromatid aberrations and acentric fragments. DIBK is non clastogenic to mammalian cells.

Conclusions:
Interpretation of results (migrated information):
negative

DIBK did not induce in increase in chromosome aberrations in rat liver cells.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

All in vitro genotoxicity studies conducted with DIBK showed clearly negative results. DIBK did not induce gene mutation in bacteria nor in mammalian cells and DIBK did also not induce chromosome aberrations in mammalian cells.

No in vivo genotoxicity studies are available with DIBK.


Short description of key information:
GLP-studies according to or equivalent to OECD guidelines 471, 473 and 476 are available for diisibutylketone.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

All in vitro genotoxicity studies conducted with DIBK showed clearly negative results. Hence, no classification for genotoxicity is applicable for DIBK.