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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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:
2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline study conducted under GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: colourless liquid

Method

Species / strain
Species / strain / cell type:
lymphocytes: human blood
Details on mammalian cell type (if applicable):
Human blood was collected aseptically from two healthy, non-smoking, adult donors, pooled (in equal volumes from each donor) and diluted with RPMI 1640 tissue culture medium supplemented with 10% foetal calf serum, 0.2 IU/mL sodium heparin, 20 IU/mL penicillin / 20 μg/mL streptomycin
and 2.0 mM L-glutamine. As lymphocytes do not normally undergo cell division, they were stimulated to do so by the addition of phytohaemagglutinin (PHA), a naturally occurring mitogen. Cultures were established from the prepared (pooled) sample and dispensed as 5 mL aliquots
(in sterile universal containers) so that each contained blood (0.4 mL), culture medium (4.5 mL) and PHA solution (0.1 mL). All cultures were then
incubated at 37°C, and the cells resuspended (at least once daily) by gentle inversion.
Metabolic activation:
with and without
Metabolic activation system:
Sprague-Dawley Male rat livers dosed with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver.
Test concentrations with justification for top dose:
In the absence of S9 mix, 3-hour treatment
2-Pentanone oxime was added to each culture in 500 μL aliquots to give final concentrations of 218.59, 364.32, 607.2 and 1012 μg/mL. Water
(purified in-house by reverse osmosis) was used as the vehicle control, and Mitomycin C at a final concentration of 0.2 μg/mL was the positive
control. Following 3-hour treatment, cultures were centrifuged at 500g for 5 minutes and the supernatant removed. Cultures were then
resuspended in saline (5 mL) and centrifuged at 500g for 5 minutes. The saline was then removed and the cell pellets resuspended in fresh
medium. They were then incubated for a further 18 hours. The cultures were then harvested, slides prepared, and microscopically examined for
cytotoxicity using a light microscope.

In the presence of S9 mix, 3-hour treatment
2-Pentanone oxime was added to each culture in 500 μL aliquots to give final concentrations of 218.59, 364.32, 607.2 and 1012 μg/mL. Water
(purified in-house by reverse osmosis) was used as the vehicle control, and Cyclophosphamide at a final concentration of 5 μg/mL was the positive control.
Following 3-hour treatment, cultures were centrifuged at 500g for 5 minutes and the supernatant removed. Cultures were then resuspended in
saline (5 mL) and centrifuged at 500g for 5 minutes. The saline was then removed and the cell pellets resuspended in fresh medium. They were then
incubated for a further 18 hours. The cultures were then harvested, slides prepared, and microscopically examined for cytotoxicity using a light
microscope.

In the absence of S9 mix, 21-hour continuous treatment
2-Pentanone oxime was added to each culture in 500 μL aliquots to give final concentrations of 218.59, 364.32, 607.2 and 1012 μg/mL. Water
(purified in-house by reverse osmosis) was used as the vehicle control, and Mitomycin C at a final concentration of 0.1 μg/mL was the positive
control.
Vehicle / solvent:
2-Pentanone oxime was soluble in water (purified in-house by reverse osmosis) at 10.12 mg/mL. On dosing a 10.12 mg/mL solution at 10% v/v into
aqueous tissue culture medium, giving a final concentration of 1012 μg/mL (10 mM), no notable culture medium changes were observed, when
compared to the vehicle control.
Controlsopen allclose all
Untreated negative controls:
no
Positive controls:
yes
Remarks:
in absence of S-9
Positive control substance:
mitomycin C
Remarks:
0.2 ug/mL (3 hr treatment); 0.1 ug/mL (21 hr treatment)
Untreated negative controls:
no
Positive controls:
yes
Remarks:
in presence of S-9
Positive control substance:
cyclophosphamide
Remarks:
5 ug/mL (3 hr treatment)
Details on test system and experimental conditions:
Vehicle control, treatment, and positive control cultures were treated approximately 48 hours after commencement of incubation of lymphocyte
cultures. Duplicate cultures were prepared throughout for each treatment (3-hour treatment in the absence and presence of S9 mix, and
21-hour continuous treatment in the absence of S9 mix). All cultures were centrifuged and resuspended in the required volume of fresh medium
before treatment, taking into account the treatment volume and S9 mix volume, where required.
In the absence of S9 mix, 3-hour treatment
2-Pentanone oxime was added to each culture in 500 μL aliquots to give final concentrations of 218.59, 364.32, 607.2 and 1012 μg/mL. Water
(purified in-house by reverse osmosis) was used as the vehicle control, and Mitomycin C at a final concentration of 0.2 μg/mL was the positive
control. After 3-hour treatment, no notable culture medium changes were observed, when compared to the vehicle control. Following 3-hour
treatment, cultures were centrifuged at 500g for 5 minutes and the supernatant removed. Cultures were then resuspended in saline (5 mL) and centrifuged at 500g for 5 minutes. The saline was then removed and the cell pellets resuspended in fresh medium. They were then incubated for a further 18 hours. The cultures were then harvested, slides prepared, and microscopically examined for cytotoxicity using a light microscope.

In the presence of S9 mix, 3-hour treatment
For treatments in the presence of S9 mix, 1 mL of S9 mix was added to give a concentration of 5% v/v in the final test medium. 2-Pentanone oxime
was added to each culture in 500 μL aliquots to give final concentrations of 218.59, 364.32, 607.2 and 1012 μg/mL. Water (purified in-house by reverse osmosis) was used as the vehicle control, and Cyclophosphamide at a final concentration of 5 μg/mL was the positive control.
After 3-hour treatment, no notable culture medium changes were observed, when compared to the vehicle control. Following 3-hour treatment, cultures were centrifuged at 500g for 5 minutes and the supernatant removed. Cultures were then resuspended in saline (5 mL) and centrifuged at
500g for 5 minutes. The saline was then removed and the cell pellets resuspended in fresh medium. They were then incubated for a further
18 hours.The cultures were then harvested, slides prepared, and microscopically examined for cytotoxicity using a light microscope.

In the absence of S9 mix, 21-hour continuous treatment
2-Pentanone oxime was added to each culture in 500 μL aliquots to give final concentrations of 218.59, 364.32, 607.2 and 1012 μg/mL. Water
(purified in-house by reverse osmosis) was used as the vehicle control, and Mitomycin C at a final concentration of 0.1 μg/mL was the positive
control. After 21-hour treatment, no notable culture medium changes were observed, when compared to the vehicle control. The cultures were then harvested, slides prepared, and microscopically examined for cytotoxicity using a light microscope.
Evaluation criteria:
The prepared slides were examined by light microscopy using a low power objective. The proportion of mitotic cells per 1000 cells in each culture
was recorded (except for positive control treated cultures, or in cultures where there were no signs of cytotoxicity).
Statistics:
The number of aberrant metaphase cells in each test substance group was compared with the vehicle control value using the one-tailed Fisher exact test (Fisher 1973).

Results and discussion

Test results
Species / strain:
lymphocytes: human donor
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
2-Pentanone oxime caused no reduction in the mitotic index at any treatment concentrationwhen compared to the vehicle control value. Therefore,
the highest three tested concentrations (364.32, 607.20 and 1012 μg/mL) were selected for metaphase analysis.

In the presence of S9 mix following 3-hour treatment, 2-Pentanone oxime caused statisticallysignificant increases (p<0.001: including gaps and
p<0.01: excluding gaps) in the proportion of metaphase figures containing chromosomal aberrations at 1012 µg/mL (a non-toxic guideline limit
concentration), when compared to the vehicle control. These increases were reproducible between duplicate cultures with mean values that exceeded the laboratory historical control range, when taken at the 99% confidence limit.

At 364.32 µg/mL, the lowest analysed concentration, a statistical increase (p<0.01: including gaps only) was observed. However, as the mean valuewas outside the 99% confidence limit yet within the laboratory historical range, with no accompanying statistical increase (excluding gaps), the
observed increase was considered biologically non-relevant. At 607.2 µg/mL, the intermediate concentration, mean values were within laboratory historical control range, when taken at the 99% confidence limit. All mean values for the vehicle control (water), were within laboratory historical control
range, when taken at the 99% confidence limit.The positive control compound, Cyclophosphamide, caused statistically significant increases
(p<0.001) in the proportion of aberrant cells. This demonstrated the efficacy of the S9 mix and the sensitivity of the test system.
As a statistically significant response was displayed at 1012 μg/mL only, in the presence of S9 mix following 3-hour treatment, with mean values that
exceeded the laboratory historical control range, no further metaphase analysis (of 21-hour continuous treatment in the absence of S9 mix) was
conducted.
No statistically significant increases in polyploid or endoreduplicated metaphases were observed during metaphase analysis, when compared to the vehicle control.

Applicant's summary and conclusion

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

2-Pentanone oxime has shown evidence of causing an increase in the frequency of structural chromosome aberrations, in the presence of S9 mix
following 3-hour treatment at 1012 μg/mL only, in this in vitro cytogenetic test system,