N-pentan-2-ylidenehydroxylamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Gene mutation in bacteria (OECD 471): negative

- Chromosome aberration test in mammalian cells (OECD 473): negative without metabolic activation, positive with metabolic activation

- Micronucleus test in mammalian cells (OECD 487): negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom

Type of assay:

bacterial reverse mutation assay

Target gene:

his- / trp-operon

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

liver preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

up to 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water (purified in house by reverse osmosis) was used as a vehicle control

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) - 1st test; preincubation- 2nd test

Evaluation criteria:

For a test to be considered valid, the mean of the vehicle control revertant colony numbers for each strain should lie within or close to the current historical control range for the laboratory unless otherwise justified by the Study Director. The historical range is maintained as a rolling record over a maximum of five years. The positive control compounds must induce an increase in mean revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537, which have relatively low spontaneous reversion rates) that of the concurrent vehicle controls. Mean viable cell counts in the 10-hour bacterial cultures must be at least 109/mL.

Statistics:

Not needed

Key result

Species / strain:

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

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

toxicity observed as a reduction of revertant colonies obtained in the Salmonella strains following exposure to 2-PO at 5000 ug/plate only in the absence of S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

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

Metabolic activation:

with

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

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

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:

All controls were within 99% of the historical range. All positive controls responded with a positive response.

Conclusions:

negative

Executive summary:

2-PO (MPKO) showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom

Type of assay:

in vitro mammalian chromosome aberration test

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 500 g for 5 minutes and the supernatant removed. Cultures were then resuspended in saline (5 mL) and centrifuged at 500 g 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 500 g for 5 minutes and the supernatant removed. Cultures were then resuspended in saline (5 mL) and centrifuged at 500 g 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.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True 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

Negative solvent / vehicle controls:

yes

True 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 500 g for 5 minutes and the supernatant removed. Cultures were then resuspended in saline (5 mL) and centrifuged at 500 g 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 500 g for 5 minutes and the supernatant removed. Cultures were then resuspended in saline (5 mL) and centrifuged at 500 g 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.

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).

Key result

Species / strain:

lymphocytes: human donor

Metabolic activation:

with

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

Key result

Species / strain:

lymphocytes: humand donor

Metabolic activation:

without

Genotoxicity:

negative

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 statistically significant 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 value was 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.

Conclusions:

negative without metabolic activation
positive with metabolic activation

Executive summary:

2-Pentanone oxime (MPKO) 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.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: Human blood

Details on mammalian cell type (if applicable):

- Type and identity of media: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)
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction, prepared from male Sprague-Dawley derived rats, dosed with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver

Test concentrations with justification for top dose:

Positive controls
In the absence of S9 mix
Identity: Mitomycin C
Solvent: Sterile purified (reverse osmosis) water
Exposure concentrations: 0.2 and 0.3 μg/mL (3 hour treatment); 0.05 and 0.1 μg/mL (20 hour treatment)

Identity: Colchicine
Solvent: Sterile purified (reverse osmosis) water
Exposure concentrations: 0.05, 0.06 and 0.07 μg/mL (3 hour treatment); 0.01, 0.02 and 0.03 μg/mL (20 hour treatment))

In the presence of S9 mix:
Identity: Cyclophosphamide
Solvent: Sterile purified (reverse osmosis) water
Exposure concentration: 5 and 10 μg/mL

Vehicle / solvent:

Vehicle(s)/solvent(s) used: The sponsor indicated that 2-Pentanone oxime was soluble in water. Water (purified in-house by reverse-osmosis) was, therefore, used as the vehicle for this study.
The highest concentration of 2-Pentanone oxime tested in this study was 10.12 mg/mL in the chosen vehicle, which provided a final concentration of 1012 μg/mL (10mM), when dosed at 10% v/v. The highest concentration in each test was diluted with water to produce a series of lower concentrations. All concentrations cited in this report are expressed in terms of the 2-Pentanone oxime as received.

The pH and osmolality of 2-Pentanone oxime in medium were tested at a concentration of 1012 ug/mL. No fluctuations in pH of the medium of more than 1 unit were observed compared with the vehicle control. No fluctuations in osmolality of the medium of more than 50 mOsm/kg were observed compared with the vehicle control.

Untreated negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

In the absence of S9 mix

Untreated negative controls:

no

Positive controls:

yes

Positive control substance:

other: Colchicine

Remarks:

In the absence of S9 mix

Untreated negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

In the presence of S9 mix

Details on test system and experimental conditions:

Lymphocyte cultures were incubated for approximately 48 hours following stimulation with PHA, before addition of the test substance. The test substance was prepared in the vehicle and dilutions made for both sets of cultures. Duplicate cultures were prepared for each treatment level and positive control cultures, quadruplicate cultures were prepared for vehicle controls. S9 homogenate was present in appropriate cultures at a final concentration of 2% v/v. All cultures were identified using unique number/colour codes. Before treatment in the absence and presence of S9 mix all cultures were re-suspended in fresh media. For cultures in the presence of S9 mix, 1 mL of medium was removed from the final volume. This was replaced with 1 mL of S9 mix immediately prior to treatment. Test substance preparations were added to cultures at 10% v/v. Cultures were incubated at 37°C for 3 hours.The cells were centrifuged and the medium was replaced with fresh medium. Cytochalasin B, at a final concentration of 6 μg/mL, was then added to all cultures. The cultures were incubated for a further 17 hours until the scheduled harvest time. Human lymphocyte cultures were set up as previously described. A 20-hour continuous treatment (1.5 to 2 normal cell cycles) at 37°C was used in the absence of S9 mix. Test substance preparations were added to cultures at 10% v/v in the presence of Cytochalasin B (6 μg/mL). The cells were harvested by centrifugation at 500 g for 5 minutes. The supernatant was removed and the cell pellet re-suspended and treated with a 4 mL hypotonic solution (0.075M KCl) at 37°C, cultures were then incubated for 3 minutes at 37°C to cause swelling. Cultures were agitated, 4 mL of ice-cold fixative (3:1 v/v methanol: acetic acid) was added slowly onto the culture surface and the cultures were slowly inverted to mix. The cultures were centrifuged at 500 g for five minutes. The supernatant was removed, and the cell pellet re-suspended. A further 4 mL of fresh fixative was then added and the cells stored at 4°C until slide preparation.

Evaluation criteria:

The following criteria were applied for assessment of assay acceptability:
Positive controls must show clear unequivocal positive responses. The negative control (solvent, vehicle control or untreated cultures) must show reproducible low and consistent micronucleus frequencies. Tests that did not fulfil the required criteria were rejected and therefore are not reported.

Statistics:

Cytotoxicity:
Cytotoxicity = 100-100{(CBPIt- 1)/(CBPIc-1)}
Where CBPI = No mononucleate cells + 2 x No binucleate cells + 3 x No multinucleate cells
Total number of cells

T = test chemical treatment culture
C = solvent control culture
Thus, a CBPI of 1 (all cells are mononucleate) is equivalent to 100% cytotoxicity.

Genotoxicity:
The analysis assumed that the replicate was the experimental unit. An arcsine transformation was used to transform the data. 2-Pentanone oxime treated groups were then compared to control using Williams’ tests (Williams 1971, 1972). The positive controls were compared to control using t-tests. Trend tests have also been carried out using linear contrasts by group number. These were repeated, removing the top dose group, until there were only 3 groups. Data were analysed using SAS 9.1.3 (SAS Institute 2002) and Quasar 1.4 (Quasar 1.4 2013).

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the absence of S9 mix, following 3-hour treatment, no significant reductions in CBPI, compared to vehicle control values, were obtained with 2-Pentanone oxime at any concentration tested. Concentrations of 2-Pentanone oxime selected for micronucleus analysis were 63.25, 126.5, 253, 506 and 1012 μg/mL.

In the absence of S9 mix following 3-hour treatment, 2-Pentanone oxime did not cause any statistically significant increases in the number of binucleate cells containing micronuclei when compared to the vehicle controls. Mean micronucleus induction in the vehicle control was within the historical control range. The positive control compounds (mitomycin C and colchicine) caused significant increases in the number of binucleate cells containing micronuclei, demonstrating the sensitivity of the test system.

In the presence of S9 mix following 3-hour treatment, a reduction in CBPI equivalent to 19% cytotoxicity, compared to vehicle control values, was obtained with 2-Pentanone oxime at 1012 μg/mL. Concentrations of 2-Pentanone oxime selected for micronucleus analysis were 63.25, 126.5, 253, 506 and 1012 μg/mL.

In the presence of S9 mix, following 3-hour treatment, 2-Pentanone oxime did not cause any statistically significant increases in the number of binucleate cells containing micronuclei when compared to the vehicle controls. Mean micronucleus induction in the vehicle control was within the historical control range. The positive control compound (Cyclophosphamide) caused a significant increase in the number of binucleate cells containing micronuclei, demonstrating the efficacy of the S9 mix and the sensitivity of the test system.

In the absence of S9 mix following 20-hour treatment, a reduction in CBPI equivalent to 55% cytotoxicity, compared to vehicle control values, was obtained with 2-Pentanone oxime at 700 μg/mL. Concentrations of 2-Pentanone oxime selected for micronucleus analysis were 50, 250, 500, 600 and 700 μg/mL.

In the absence of S9 mix, following 20-hour treatment, 2-Pentanone oxime did not cause any statistically significant increases in the number of binucleate cells containing micronuclei when compared to the vehicle controls. Mean micronucleus induction in the vehicle control was within the historical control range.The positive control compounds (mitomycin C and colchicine) caused a significant increase in the number of binucleate cells containing micronuclei, demonstrating the sensitivity of the test system.

Conclusions:

negative with and without activation

Executive summary:

It was concluded that 2-Pentanone oxime (MPKO) administered for 3 hours in both the absence and presence of S9 mix and for 20 hours in the absence of S9 mix only, did not show any evidence of causing an increase in the induction of micronuclei in cultured human lymphocytes, in this in vitro test system under the experimental conditions described.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

- Bone marrow chromosome aberration test (OECD 475): negative

- Comet assay (similar to OECD 489): negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

GLP compliance:

yes (incl. QA statement)

Remarks:

Health Care Inspectorate, Ministry of Health, Welfare and Sport, Den Haag, The Netherlands

Type of assay:

mammalian bone marrow chromosome aberration test

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: colony maintained under specific pathogen-free (SPF) conditions at Charles River (Sulzfeld, Germany)
- Age at study initiation: 8 weeks
- Weight at study initiation: mean body weights: 328 g
- Housing: under conventional conditions in Makrolon® cages (type IV) with a bedding of wood shavings (Lignocel, Rettenmaier & Söhne GmbH & Co, Rosenberg, Germany) and strips of paper (Enviro-dri, Shepherd Specialty Papers, Michigan, USA) and a wooden block (ABEDD, Vienna, Austria) as environmental enrichment
- Diet (e.g. ad libitum): cereal-based rodent diet (Rat & Mouse No. 3 Breeding Diet, RM3)
ad libitum from the arrival of the animals until the end of the study, except during inhalation exposure and during the fasting period prior to the collection of blood for clinical pathology
- Water (e.g. ad libitum): domestic mains tap-water suitable for human consumption (quality guidelines according to Dutch legislation based on EC Council Directive 98/83/EC). The water was given in polypropylene bottles, which were cleaned weekly and filled as needed.
ad libitum from the arrival of the animals until the end of the study, except during inhalation exposure and during the fasting period prior to the collection of blood for clinical pathology
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 45 - 65%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): Lighting was artificial (fluorescent tubes) with a sequence of 12 hours light and 12 hours dark.

IN-LIFE DATES: From: 2014-01-22 To: 2014-02-05

Route of administration:

inhalation: vapour

Vehicle:

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

Details on exposure:

TYPE OF INHALATION EXPOSURE: nose only
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: nose-only inhalation chambers (each group in a separate chamber; chamber types; groups 2-4: a modification of the chamber manufactured by ADG Developments Ltd., Codicote, Hitchin, Herts, SG4 8UB, United Kingdom; group 1: chamber manufactured by P. Groenendijk Kunststoffen B.V., the Netherlands; see Figure 1). The inhalation chamber consisted of a cylindrical column of aluminum (groups 2-4) or polypropylene (group 1), surrounded by a transparent cylinder.
- Volume column: 39 (group 1) or 37 litres (groups 2-4)
- Details column: top assembly with the entrance of the unit, one mixing chamber, a rodent tube section, and at the bottom the base assembly with the exhaust port.
- Method of holding animals in test chamber: The animals were secured in plastic animal holders (Battelle), positioned radially through the outer cylinder around the central column.
- Airflow through unit: at least 1 litre/min per animal
- Temperature, humidity, pressure in air chamber: The chamber airflow of the test atmospheres was recorded about hourly by means of the settings of the flow controllers. The temperature and the relative humidity of the test atmospheres were measured continuously and recorded every minute using a CAN transmitter with temperature and relative humidity probes (G.Lufft Mess- und Regeltechnik GmbH, 70719 Fellbach, Germany). The concentrations of oxygen (oxygen analyser type PMA-10, M&C Products Analysentechnik GmbH, Ratingen-Lintorf, Germany) and carbon dioxide (GM70, Vaisala, Helsinki, Finland) in the test atmosphere were measured during exposure on the first exposure day.

Duration of treatment / exposure:

10 days

Frequency of treatment:

6 hours/day - 5days/week

Dose / conc.:

52.9 ppm (analytical)

Remarks:

+/- 2.8 ppm

Dose / conc.:

149.3 ppm (analytical)

Remarks:

+/- 1.9 ppm

Dose / conc.:

298.9 ppm (analytical)

Remarks:

+/- 3.1 ppm

No. of animals per sex per dose:

5

Control animals:

yes

Positive control(s):

- Substance: mutagen mitomycin-C (MMC)
- Concentration: 3 mg/kg bw
- Treatment: once intraperitoneal (stock concentration 0.3 mg/ml in physiological saline; dosing volume 10 ml/kg-bw; dose level 3.0 mg/kg bw)
- Time: ca. 24 h before sacrifice

Tissues and cell types examined:

Bone marrow cells

Details of tissue and slide preparation:

Administration of Colchicine:
Ca. 3 h prior to sacrifice, all animals were injected intraperitoneally with colchicine (stock concentration 0.4 mg/mL in physiological saline; dosing volume 10 mL/kg-bw; dose level 4 mg/kg bw) to accumulate metaphase cells in the bone marrow.
Animals were sacrificed by decapitation under CO2/O2 anaesthesia.

Bone marrow collection and processing
24 h after the last exposure, animals were sacrificed for isolation of bone marrow in a random order. The animals of the positive control group were sacrificed ca. 24 h after intraperitoneal dosing with MMC. Following sacrifice, the bone marrow cells of both femurs were collected into Hank’s balanced salt solution (HBSS), treated with a hypotonic solution (0.075 M potassium chloride), fixed with a freshly prepared 3:1 (v/v) mixture of methanol and acetic acid and processed for chromosome preparations. Four slides were prepared for each animal. Slides were stained with Giemsa, air-dried and mounted with a coverslip. Remaining cell suspensions were kept refrigerated until it had been confirmed that a sufficient number of cells were present on the slides prepared. If necessary, additional slides could have been prepared from the remaining cell suspension.

The slides were randomly coded by a person not involved in the scoring to enable ‘blind’ scoring. Slides (two per animal for mitotic index and four per animal for aberration analysis) were read by moving from the beginning of the slide (label end) to the leading edge in horizontal lines taking care that areas selected for evaluation were evenly distributed over the whole slide. If feasible, 1000 cells (500 on each of the two slides) were examined in each animal to determine the percentage of cells in mitosis (mitotic index). Of each animal, 200 well-spread metaphases (50 metaphases per slide), each containing 40-42 centromeres, were analysed by microscopic examination for chromatid- type aberrations, chromosome-type aberrations, and other anomalies. If heavily damaged or endoreduplicated cells were observed, this was recorded but the cells were not counted and not included in the 200 analysed cells. The frequency of polyploid cells was recorded when observed. The Vernier readings of all aberrant metaphases were recorded.

Evaluation criteria:

The study was considered valid if the positive control group gives a statistically significantly increase in the percentage of cells with structural chromosomal aberrations when compared to the percentage found in the negative control group and if the percentage of aberrant cells found in the negative control group is within the historical range.
The criteria for determining a positive result are a dose-related increase in the relative number of cells with structural chromosomal aberrations or a clear increase in the number of cells with structural chromosomal aberrations in a single dose group (at a single sampling time). Positive results from the in vivo chromosomal aberration test indicate that a test material induces structural chromosomal aberrations in the bone marrow of the species tested.
A test material is considered to be negative if it produces no statistically significant increase in the number of cells with structural chromosomal aberrations at any of the dose levels analysed. Negative results indicate that a test material does not induce structural chromosomal aberrations in the bone marrow of the species tested.
Biological relevance of the results should be considered first. Statistical methods may be used as an aid in evaluating the test results.

Statistics:

Statistical tests were performed using GraphPad Prism®, Version 5.03, Copyright © 1992-2010 GraphPad Software, Inc., CA, USA.
In all tests a significance level of 5% was used (α = 0.05).
Data on percentage of cells with aberrations and mitotic index were analysed by one-way analysis of variance [ANOVA]. Two ANOVA models were applied. In one of the ANOVA models it was tested if the positive control differed from the negative control (t-test). In the other ANOVA model (including Dunnett’s test as post-hoc test) it was tested if the test material (different doses) differed from the negative control. It was checked if the ANOVA assumptions were met (i.e. if variances are equal). In case assumptions were not met, square root transformation (sqrt(x+1)) was applied to 'normalise' the distribution of the counts. In case this was not sufficient, non-parametric testing was performed using the Mann-Whitney test (positive control compared with negative control) or Kruskal-Wallis analysis of variance (test material groups compared with negative control).

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Slide analysis revealed that three animals (no. 20: group 1; no. 36 and no. 42: both group 2) showed a very low number of metaphases, demonstrated by the very low mitotic index. The number of metaphases was too small for appropriate analysis of aberrations. It was decided to exclude all three animals from further evaluation. The data presented in Table 1 (section 'any other information on results') are the group means excluding these animals.
To compensate for the loss of two animals in group 2, a spare animal was included (animal no. 34). As a result, group 1 and 2 contained only four instead of five animals. Although this is lower than the required number of animals according to the OECD guideline 475, the numbers of data collected in combination with the low inter-animal variability within the groups are considered sufficient to draw unambiguous conclusions.

The results of the rats treated with the positive control substance MMC (group 5) showed the expected statistically significant increase (p<0.01) of cells with structural chromosome aberrations (including and excluding cells with only gaps), when compared to the negative control (clean air: group 1). The mean mitotic index was reduced to 52% of the mean mitotic index of the negative control group (clean air). These results demonstrate that the positive control substance MMC reached the bone marrow and induced damage to the chromosomes of the bone marrow cells of male rats.

The number of structural chromosomal aberrations found in the negative control group was within the historical range. These results demonstrated the validity of the test system.

The number of cells with structural chromosome aberrations in the rats treated with the test material, MPKO, were comparable to the number of cells with structural chromosome aberrations found in the concurrent negative control (clean air) and did not demonstrate a statistically significant increase. The test material, MPKO, induced a statistically significant decrease in the mean mitotic index (80%), when compared to the mean mitotic index of the concurrent vehicle control group (100%). These results indicate that the test material reached the bone marrow by the general circulation. No statistically significant difference in the mean mitotic index was observed when comparing the animals of group 2 and 3 to the concurrent negative control animals, which reflects a lack of toxic effects on erythropoiesis at the lowest two concentration levels of the test material.

Table 1: Group mean percentage of cells with aberrations +/- SD (including and excluding cells with only gaps) and mitotic index after inhalation exposure to 2 -PO

² Mann-Whitney p-value: 0.0090 (incl. gaps) or 0.0095 (excl. gaps)

3 Dunnett’s test p-value:<0.05
4 Unpaired t-test p-value: <0.0001

Group	Target concentration in air (ppm)	% cells with aberrations(mean +/-SD)  incl. gaps excl. gaps	Mitotic index  mean +/- SD Relative (%)
1. Negative control	0	0.6 +/-0.3 0.3 +/-0.3	5.0 +/-0.5 100
2. Low	50	0.6 +/-0.3 0.1 +/-0.3	4.4 +/-0.7  89
3. Mid	150	0.1 +/-0.2  0.0 +/-0.0	4.3 +/-0.4 87
4. High	300	0.6 +/-0.2 0.2 +/-0.3	4.0^3 +/-0.5 80^3
5. Positive control	Mitomycin-C	22.8 +/-8.4^2 22.3 +/-8.1^2	2.6^4 +/-0.4  52^4

Conclusions:

It is concluded that, under the conditions used in this study, 2-PO, at concentrations up to 298.9 ppm (actual concentration; 6 hours/day, 5 days/week), did not induce chromosome aberrations in the bone marrow of male rats. The target concentration of 300 ppm (actual concentration of 298.9 ppm) was selected just below the maximum saturated vapor concentration for 2-PO.

Reference 2

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: Tice et al. 2000 and Hartmann et al. 2003

Deviations:

yes

Remarks:

Single cells were erroneously suspended in 0.5% (w/v) normal melting agarose solution in phosphate buffered saline (PBS) instead of 0.5% (w/v) low melting agarose solution in PBS. No abnormalities were observed on the slides.

Principles of method if other than guideline:

The protocol of the study is in agreement with the recently developed OECD guideline 489 (In Vivo Mammalian Alkaline Comet Assay; 2014/09/26), since this guideline is based on the references listed here below, which were used in this study.
Tice RR, Agurell E, Anderson D, Burlinson B, Hartmann A, Kobayashi H, Miyamoe Y, Rojas E, Ryu JC, Sasaki F (2000). Single cell gel/comet assay: guidelines for an in vitro and in vivo genetic toxicology testing. Environmental and molecular mutagenesis; vol 35; pp 206-221.
Hartmann A, Agurell E, Beevers C, Brendler-Schaab S, Burlinson B, Clay P, Collins A, Smith A, Speit G, Thybaud V, Tice RR (2003). Recommendations for conducting the in vivo alkaline Comet assay. Mutagenesis; vol 18; no 1; pp 45-51.

GLP compliance:

yes (incl. QA statement)

Remarks:

Health Care Inspectorate, Ministry of Health, Welfare and Sport, Den Haag, The Netherlands

Type of assay:

mammalian comet assay

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: colony maintained under specific pathogen-free (SPF) conditions at Charles River (Sulzfeld, Germany)
- Age at study initiation: 8 weeks
- Weight at study initiation: mean body weights: 328 g for male and 205 g for female animals
- Housing: under conventional conditions separated by sex, in Makrolon® cages (type IV) with a bedding of wood shavings (Lignocel, Rettenmaier & Söhne GmbH & Co, Rosenberg, Germany) and strips of paper (Enviro-dri, Shepherd Specialty Papers, Michigan, USA) and a wooden block (ABEDD, Vienna, Austria) as environmental enrichment
- Diet (e.g. ad libitum): cereal-based rodent diet (Rat & Mouse No. 3 Breeding Diet, RM3)
ad libitum from the arrival of the animals until the end of the study, except during inhalation exposure and during the fasting period prior to the collection of blood for clinical pathology
- Water (e.g. ad libitum): domestic mains tap-water suitable for human consumption (quality guidelines according to Dutch legislation based on EC Council Directive 98/83/EC). The water was given in polypropylene bottles, which were cleaned weekly and filled as needed.
ad libitum from the arrival of the animals until the end of the study, except during inhalation exposure and during the fasting period prior to the collection of blood for clinical pathology
- Acclimation period: 7 days

IN-LIFE DATES: From: 2014-01-22--24 To: 2014-02-05--07 depending on the groups.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 45 - 65%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): Lighting was artificial (fluorescent tubes) with a sequence of 12 hours light and 12 hours dark.

Route of administration:

inhalation: vapour

Vehicle:

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

Details on exposure:

EXPOSURE APPARATUS:
nose-only inhalation chambers (each group in a separate chamber; chamber types; groups 2-4: a modification of the chamber manufactured by ADG Developments Ltd., Codicote, Hitchin, Herts, SG4 8UB, United Kingdom; group 1: chamber manufactured by P. Groenendijk Kunststoffen B.V., the Netherlands; see Figure 1). The inhalation chamber consisted of a cylindrical column of aluminum (groups 2-4) or polypropylene (group 1), surrounded by a transparent cylinder.
VOLUME COLUMN: 39 (group 1) or 37 litres (groups 2-4)
DETAILS COLUMN: top assembly with the entrance of the unit, one mixing chamber, a rodent tube section, and at the bottom the base assembly with the exhaust port.
METHOD OF HOLDING ANIMALS IN TEST CHAMBER:
The animals were secured in plastic animal holders (Battelle), positioned radially through the outer cylinder around the central column.
TOTAL AIRFLOW THROUGH UNIT:
at least 1 litre/min per animal

GENERATION TEST ATMOSPHERE:
The inhalation equipment was designed to expose rats to a continuous supply of fresh test atmosphere. To generate the test atmospheres, a liquid flow of test material, controlled by a motor driven syringe pump (WPI Type SP220i, World Precision Instruments, Sarasota FL, USA), was allowed to evaporate in a mass flow controlled stream of humidified air, by directing it through a glass vaporator at 65.0 ̊C. The resulting atmosphere was cooled by leading it through a coil condenser which was controlled at 19 ̊C. The vapour was transported in a stream of humidified compressed air, the flow of which was controlled by means of a mass flow controller (Bronkhorst, Hi Tec, Ruurlo, The Netherlands).
The test atmospheres for the mid concentration and high concentration groups were generated separately (i.e. by using a separate syringe pump, evaporator and condensor for these concentrations). The test atmosphere for the low concentration group was obtained by diluting the high-concentration test atmosphere. For this purpose, a mass flow controlled stream of the high-concentration atmosphere was supplemented with a mass flow controlled stream of humidified compressed air via an eductor (Fox Eductor from Fox Valve Development Corp., Dover, NJ, USA). Each test atmosphere was directed to the top inlet of an exposure unit, led to the noses of the animals and exhausted at the bottom of the unit.
The exposure unit for the control animals was supplied with a measured stream of humidified compressed air only.
The animals were placed in the exposure unit after stabilization of the test atmosphere.

TEMPERATURE, HUMUDITY, PRESSURE IN AIR CHAMBER: The chamber airflow of the test atmospheres was recorded about hourly by means of the settings of the flow controllers. The temperature and the relative humidity of the test atmospheres were measured continuously and recorded every minute using a CAN transmitter with temperature and relative humidity probes (G.Lufft Mess- und Regeltechnik GmbH, 70719 Fellbach, Germany). The concentrations of oxygen (oxygen analyser type PMA-10, M&C Products Analysentechnik GmbH, Ratingen-Lintorf, Germany) and carbon dioxide (GM70, Vaisala, Helsinki, Finland) in the test atmosphere were measured during exposure on the first exposure day.

Duration of treatment / exposure:

10 days

Frequency of treatment:

6 hours/day -- 5days/week
On the day of sacrifice, the animals were exposed for an additional 2 h period.

Dose / conc.:

52.9 ppm (analytical)

Remarks:

+/- 2.8 ppm

Dose / conc.:

149.3 ppm (analytical)

Remarks:

+/- 1.9 ppm

Dose / conc.:

298.9 ppm (analytical)

Remarks:

+/- 3.1 ppm

No. of animals per sex per dose:

5

Control animals:

yes

Positive control(s):

- Substance: 2-acetylaminofluorene (2-AAF)
- Concentration: 50 mg/kg bw
- Treatment: by gavage (50 mg/kg bw, 2.5 mg/mL in corn oil, dosing volume 20 mL/kg)
- Time: administered 12-16 h before sacrifice

Tissues and cell types examined:

Hepatocytes

Details of tissue and slide preparation:

Experimental procedure
Hepatocytes were obtained by liver perfusion. Since only a limited number of animals could be handled for liver perfusion in one day, the animals of groups 1, 2, 3 and 4 were necropsied on three consecutive days in a stratified random order. On the day before sacrifice the exposure terminated later than on the previous exposure days to represent the late sampling time (i.e. to represent the effects 16-26 h after exposure). The 2-h exposure period on the day of sacrifice was included to represent the effects after the short sampling time (i.e. 2-6 h after exposure). All animals of the positive control group were necropsied on the same day. Reserve rat no. 12 (group 1) was used to replace rat no. 8, because the liver perfusion was not appropriate (no cells were obtained). Hepatocytes of reserve rats 56 (group 3) and 110 (group 6) were isolated to replace any rat of these groups, if required. Eventually, slides of these animals were not used. No hepatocytes were isolated from reserve rats 34 and 78.

Isolation of hepatocytes
After the last exposure, the animals were sacrificed for isolation of hepatocytes in a stratified random order. The animals of the positive control group were sacrificed 12-16 h after oral dosing. Hepatocytes were isolated from the liver using the perfusion technique described by Williams et al. (1977) with minor modifications. Briefly, the liver of each rat was perfused in situ with a 0.01 M HEPES buffer whilst under sodium pentobarbital anaesthesia and exsanguination from the abdominal aorta, followed by an in vitro perfusion with a 0.1 M HEPES-buffered collagenase solution.
Directly after perfusion, a small part of the caudate lobe was tied off using a ligature. Subsequently, part of the lobe was removed and preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde (10 % solution of formalin) for histopathological examination (see section 4.11.7 of the study report). After isolation, the dissociated cells were incubated in a shaking water bath whereafter, they were filtered, centrifuged and resuspended in Williams medium E. Cell counts were made and the viability of the hepatocytes was determined by trypan blue exclusion.

Preparation of slides
Microscopic slides were prepared by mixing an aliquot of the cell suspension with a normal- melting agarose solution (0.5 % (w/v) in PBS), this mixture was loaded on a glass slide, pre-coated with normal-melting agarose (1.5 % (w/v) in PBS), and mounted with a coverslip. Three slides per animal were prepared. The slides were stored on a cold plate until the agarose had solidified. Subsequently, the coverslip was removed and the slide was incubated in lysis buffer (2.5 M NaCl, 0.1 M Na2EDTA, 0.175 M NaOH, 0.01 M Tris in Milli-Q water, supplemented with 1 % Triton X-100 (v/v), pH 10) overnight. Subsequently, slides were incubated in cold electrophoresis buffer (0.3 M NaOH, 0.001 M Na2EDTA in Milli-Q water, pH >13) for 30 ± 1 min, followed by electrophoresis (ca. 25 V and 300 mA) for 30 ± 1 min in cold electrophoresis buffer, while cooled on ice. After incubation in neutralization buffer (0.4 M Tris in Milli-Q water, pH 7.5), slides were dehydrated by incubating in ethanol at room temperature and air-dried.

Slides were coded by a person not involved in analysing to enable ‘blind’ scoring. Slides were stained with ethidium bromide solution (20 μg/mL in Milli-Q water) which was directly pipetted on the slide and covered with a coverslip just before analysis. A fluorescent microscope connected to a camera and Comet Assay IV software (Perceptive Instruments) was used for the analysis of the slides. Fifty cells (randomly selected starting from the center of the slide) per slide and three slides per animal were analysed. Ghost cells, with a small head and a diffuse and large tail, were excluded from analysis, but their presence was counted as an indication of cytotoxicity.

Evaluation criteria:

The study is considered valid if the group mean tail intensity of the positive control group gives a statistically significant increase compared to the group mean of the negative control group and if the group mean tail intensity of the negative control is within the historical range.
A test material is considered to be positive in the in vivo comet assay if a statistically significant increase is observed at one or more dose levels compared to the group mean of the negative control group and/or if a significant dose related increase in the group mean tail intensity is observed. Positive results indicate that the test material has the potential to induce primary DNA damage in vivo in the tissue evaluated, under the conditions used in this study.
A test material is considered to be negative if no statistically significant increase is observed at any of the dose levels compared to the group mean of the negative control group. Negative results indicate that the test material does not have the potential to induce DNA damage in vivo in the tissue evaluated, under the test conditions used in this study.
Biological relevance was taken into account for interpretation of the results. Statistical methods were used as an aid for interpretation of the results.

Statistics:

The median tail intensity (%tail DNA) was calculated per slide, followed by calculation of the mean of the three medians per animal and calculation of the group mean.
Statistical tests were performed using GraphPad Prism®, Version 5.03, Copyright © 1992-2010 GraphPad Software, Inc., CA, USA.
In all tests a significance level of 5% was used (α = 0.05).
Data on group mean tail intensity were analysed by one-way analysis of variance (ANOVA). Two ANOVA models were applied. In the first ANOVA model it was tested if the positive control differed from the negative control (t-test). In a second ANOVA model (including Dunnett’s test as post-hoc test) it was tested if the test material (different doses) differed from the negative control. It was checked if the ANOVA assumptions were met (i.e. if variances were equal). In case assumptions were not met, non-parametric testing was performed using the Mann-Whitney test (positive control compared with negative control) or Kruskal-Wallis analysis of variance (test material groups compared with negative control).

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The group mean tail intensity and percentage of ghost cells are presented in Table 1 (section 'any other information on results).
The positive control substance 2-AAF demonstrated a statistically significant increase in tail intensity compared to the negative control group (p < 0.05). Mean tail intensity of the negative control group was slightly higher than the maximum value of the historical range. However, the historical data included a limited number of animals. In this study the tail intensity of the negative control was considered sufficiently low to reliably detect the potential of the test material to induce primary DNA damage. Therefore, the comet assay performed in this study was considered valid.

The percentage of ghost cells on the slides of the test material groups and the positive control group were comparable to the percentage of ghost cells observed on the negative control group. Group mean tail intensity of the test material MPKO was comparable to the negative control group and did not demonstrate a statistically significant increase in tail intensity compared to the negative control group at any of the concentrations tested.

Table 1 Group mean tail intensity and percentage of ghost cells  SD in isolated male rat hepatocytes after exposure to 2 -PO

Group	Target concentration in air (ppm)	Tail intensity (%tail DNA)(mean +/- SD)	Percentage ghost cells(mean +/- SD)
1. Negative control	0	3.2 +/-1.3	12 +/-5
2. Low	50	3.2 +/-1.5	16 +/-8
3. Mid	150	5.1 +/-0.9	12 +/-3
4. High	300	3.8 +/-1.3	12 +/-4
5. Positive control	2 -AAF	17.3 +/-5.6*	10 +/-2

* Mann-Whitney p-value: 0.0079

Conclusions:

It is concluded that, under the conditions used in this study, the test material 2-PO, at concentrations up to 298.9 ppm (actual concentration; 6 hours/day, 5 days/week), did not induce primary DNA damage to hepatocytes of male rats. The target concentration of 300 ppm (actual concentration of 298.9 ppm) was selected just below the maximum saturated vapor concentration for 2-PO.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro

 

In vitro gene mutation study in bacteria

2-pentanone oxime (2-PO; CAS623-40-5) was investigated for mutagenicity in bacteria (Ames test) in a study according to OECD Guideline 471 under GLP conditions (Key, 2013, Ames). The Salmonella typhimurium strains TA 1535, TA 1537, TA 100 and TA 98 and the Escherichia coli WP2 uvr A/pKM101 were exposed to concentrations up to 5000 µg/plate in water with and without the addition of a metabolic activation system (S9 mix), respectively, in two independent experiments (plate incorporation and preincubation method). 2-PO did not induce gene mutations in bacteria under the conditions of the test. Cytotoxicity was only observed (as reduction of revertant colonies obtained) in the Salmonella strains following exposure to 2-PO at 5000 µg/plate only in the absence of S9 mix. The positive controls and the vehicle controls were valid, demonstrating that the study had been performed properly. In conclusion, 2-PO was found to have no bacterial reverse mutagenicity.

In vitro chromosome aberration study in mammalian cells

A GLP conform chromosome aberration study was performed according to OECD Guideline 473 (Key, 2013, CA). Clastogenicity of 2-PO (CAS 623-40-5)was investigated in human blood lymphocytes. Cells were treated with the test material both with and without the addition of cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from livers of rats treated with phenobarbital and 5,6-benzoflavone. The following concentrations were tested: 218.59, 364.32, 607.2 and 1012 μg/mL with and without metabolic activation. Cultures of all concentrations were exposed to the test substance for 3 h followed by harvesting after 18 h. In addition, without metabolic activation cells were continuously treated for 21 h. The positive controls used were mitomycin C in the absence and cyclophosphamide in the presence of metabolic activation. Duplicate cultures were tested for every test and positive and solvent controls were set up and handled in parallel. The prepared slides were examined by light microscopy using. 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). Cytotoxicity was determined using a light microscope. The number of aberrant metaphase cells in each test substance group was compared with the vehicle control value. 2-PO caused no reduction in the mitotic index at any treatment concentration when 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 h treatment, 2-PO caused statistically significant 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 lowestanalysedconcentration, a statistical increase (p<0.01: including gaps only) was observed. However, as the mean value was 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 h treatment, with mean values that exceeded the laboratory historical control range, no further metaphase analysis (of 21 h 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.Thus,2-PO has shown evidence of causing an increase in the frequency of structural chromosome aberrations, in the presence of S9 mix following 3 h treatment at 1012 μg/mL, in this in vitro cytogenetic test system.

In vitro micronucleus test

In a GLP and OECD Guideline 487 compliant study 2-PO (CAS 623-40-5)was tested in an in vitro micronucleus test using human blood lymphocytes (Key, 2013, MN). Lymphocyte cultures were incubated for approximately 48 h following stimulation with phytohaemagglutinin (PHA), before addition of the test substance. The test substance was prepared in the vehicle (water) to reach the final test concentration. The test was carried out employing 2 exposure times without S9 mix: 3 and 20 h, and 1 exposure time with S9 mix: 4 h. The harvesting time was either 17 h after the 3 h exposure and directly after the 20 h exposure. The incidence of mononucleate, binucleate and polynucleate cells per 500 cells was assessed per culture. The presence of an unusual number of, for example, cells undergoing mitosis, polyploid cells, necrotic cells and debris was also noted. From these results, concentrations were selected for micronucleus analysis. The highest concentration was intended to be that which caused a depression in the cytokinesis-block proliferative index (CBPI) equivalent to 55 +/- 5% cytotoxicity (approximately) when compared with the concurrent vehicle control. Interphase cells were examined by fluorescence microscopy and the incidence of micronucleated cells per 1000 binucleate cells per culture were scored where possible.In the absence of S9 mix, following 3 h treatment, no significant reductions in CBPI, compared to vehicle control values, were obtained with 2-PO at any concentration tested. In the presence of S9 mix, following 3 h treatment, a reduction in CBPI equivalent to 19% cytotoxicity, compared to vehicle control values, was obtained with 2-PO at 1012 μg/mL. Concentrations of 2-PO selected for micronucleus analysis for both experiments were 63.25, 126.5, 253, 506 and 1012 μg/mL. 2-PO did not cause any statistically significant increases in the number of binucleate cells containing micronuclei when compared to the vehicle controls. Mean micronucleus induction in the vehicle control was within the historical control range. The positive control compounds (-S9: mitomycin C and colchicine; +S9: cyclophosphamide) caused significant increases in the number of binucleate cells containing micronuclei, demonstrating the sensitivity of the test system. In the absence of S9 mix following 20 h treatment, a reduction in CBPI equivalent to 55% cytotoxicity, compared to vehicle control values, was obtained with 2-PO at 700 μg/mL. Concentrations of 2-PO selected for micronucleus analysis were therefore 50, 250, 500, 600 and 700 μg/mL. Also here, no statistically significant increases in the number of binucleate cells containing micronuclei were identified. The positive control compounds (mitomycin C and colchicine) were reliable. Thus, 2-PO revealed no indications of chromosomal damage in the in vitro micronucleus test in the absence and in the presence of metabolic activation, under the conducted test conditions.

In vivo

In vivo bone marrow chromosome aberration study

Genotoxicity in vivo of 2-pentanone oxime (2-PO; CAS 623-40-5) was assessed by a chromosome aberration test according to the OECD Guideline 475 and GLP compliance (Key, 2014, CA). The objective of the chromosome aberration test was to examine the potential of the test material 2-PO to cause damage to the chromosomes of bone marrow cells by analysis of metaphases as sampled in bone marrow of male rats after inhalation exposure of the test material for 6 h/day, 5 days/week over a 14-day period (total of 10 exposure days). Five groups of five male Sprague-Dawley rats were included in the test, a positive control group (treated with mitomycin C), a negative control group and three groups exposed nose-only to target concentrations of 50, 150 and 300 ppm (analytical concentration: 52.9 (± 2.8), 149.3 (± 1.9) and 298.9 (± 3.1) ppm) for 6 h/day, 5 days/week, during 2 consecutive weeks. The target concentration for the high concentration group was selected just below the saturated vapour concentration. Inhalation exposure to 2-PO did not induce chromosomal damage in bone marrow cells, under the conditions of this study. The results of the positive and negative control groups were in the expected ranges. Based on the measurement of 2-PO and its metabolites in the blood of rats after a single (6-h) exposure by inhalation within the sub-chronic inhalation toxicity study (please refer to the technical dossier, section 7.1 Toxicokinetics for more details), it is concluded that 2-PO has reached the blood, and therefore the systemic exposure has occurred at the highest attainable exposure condition. This implies that the in vivo chromosome aberration test is valid.

Comet assay

Genotoxicity in vivo of 2-PO (CAS 623-40-5) was assessed by the comet assay, according to the recommendations of international experts (e.g. Tice et al., 2000 and Hartmann et al., 2003) and GLP compliance (Key, 2014, Comet). The objective of this in vivo comet assay was to examine the potential of the test material 2-PO to cause primary DNA damage, such as single and double strand DNA breaks, alkali labile sites and incomplete repair sites, in hepatocytes of rats after inhalation exposure to the test material for 6 h/day, 5 days/week over a 14-day period (total of 10 exposure days). Four main groups of five male were exposed nose-only to target concentrations of 0 (control), 50, 150 and 300 ppm of the test material. The target concentration for the high concentration group was selected just below the saturated vapour concentration. The male rats of the main groups were additionally exposed during a 2 h period just before their scheduled sacrifice. In addition, the study included a positive control group (six male rats treated with 2-acetylaminofluorene, 2-AAF).

Inhalation exposure to 2-PO did not induce primary DNA damage in hepatocytes in male rats under the conditions of this study. The results of the positive and negative control groups were in the expected ranges, indicating that the in vivo genotoxicity assays were valid. The results of the positive and negative control groups were in the expected ranges. Based on the measurement of 2-PO and its metabolites in the blood of rats after a single (6-h) exposure by inhalation within the sub-chronic inhalation toxicity study (please refer to the technical dossier, section 7.1 Toxicokinetics for more details), it is concluded that 2-PO has reached the blood, and therefore the systemic exposure has occurred at the highest attainable exposure condition.

Conclusion

Overall,2-PO (CAS 623-40-5) was considered to be not mutagenic in bacterial cells with and without metabolic activation. 2-PO did not induce micronuclei with and without metabolic activation and was not clastogenicin the absence of a metabolic activation system in cultured human blood lymphocytes. However, in the presence of a metabolic activation system 2-PO caused an increase in the frequency of structural chromosome aberrations in human blood lymphocytes. As this positive result was not confirmed in an in vivo chromosomal aberration test with bone marrow cells and 2-PO did also not induce primary DNA damage in an in vivo comet assay with rat hepatocytes, 2-PO is considered as non-mutagenic and non-clastogenic in vivo.

Justification for classification or non-classification

The available data on genetic toxicity do not meet the criteria for classification according to Regulation (EC) 1272/2008 and are therefore conclusive but not sufficient for classification.