N-pentan-2-ylidenehydroxylamine

Genetic toxicity: in vivo

Administrative data

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

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting 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

Objective of study:

distribution

Qualifier:

according to guideline

Guideline:

other: modified OECD 412

Deviations:

not applicable

Principles of method if other than guideline:

Blood levels of 2-pentanone oxime (2-PO) and its metabolite methyl propyl ketone (MPK) were measured with inhalation exposure in a GLP study in which 5 male rats were exposed nose-only to the oxime at an average concentration of 297 ppm. Blood samples for analysis were collected prior to exposure, at 2, 4, and 6 hours during the exposure and 6 hours following termination of the exposure. Analysis was performed using a validated method.

GLP compliance:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 8 weeks old
- Weight at study initiation: 351 g
- Housing: individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2
- Humidity (%): 45-65
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 hrs

Route of administration:

inhalation: vapour

Vehicle:

other: air

Details on exposure:

TYPE OF INHALATION EXPOSURE: nose only

Duration and frequency of treatment / exposure:

6 hours

Dose / conc.:

297 ppm

No. of animals per sex per dose / concentration:

5

Control animals:

other: Blood samples were collected prior to exposure. This data is considered as control data.

Details on study design:

- Observation period: prior to exposure, 2, 4 and 6 hours of exposure period

Details on dosing and sampling:

Blood samples for analysis were collected prior to exposure, at 2, 4, and 6 hours during the exposure and 6 hours following termination of the exposure. Chemical analysis of the blood samples was used to determine the concentration in blood of 2-PO and its metabolite MPK. Analysis was performed using a validated method. Briefly, the method of analysis was Headspace Gas Chromatography (HS-GC) with Mass Spectrometry (MS) and a Combipal autosampler, on a DB1701 column, splitless injection and a temperature gradient (0 °C (5 min.) →10 °C/min → 280°C (2min)) and Helium as carrier gas.

Details on metabolites:

Both detectable levels of 2-PO and MPK were present following 2 hours of exposure (average 10.2 and 1.3 mg/kg blood, respectively) and concentrations increased during the 6-hour exposure period and rapidly declined following exposure such that detectable levels [LOD (limit of detection) = 2 mg/kg blood] were no longer present 6 hours after the termination of the exposure. These results indicate that 2-pentanone oxime reaches the blood with inhalation producing systemic exposure.

	Hours of exposure	Measured 2-PO concentration (mg/kg)	Measured MPK concentration (mg/kg)
average	0 (predose)	<LOD	<LOD
SD	0 (predose)	NA	NA
average	2	10.0	1.3
SD	2	1.1	0.1
average	4	17.7	2.6
SD	4	0.6	0.5
average	6	>ULOQ *	4.2
SD	6	NA	1.3
average	6 h post exposure	<LOD	<LOD

NA- Not Appropriate; LOD- Limit of Detection; ULOQ- Upper Limit of Quantification

* LOD is 2 mg/kg; LLOQ 4.4 mg/kg for 2 -PO and 4.2 mg/kg for MPK

Conclusions:

no bioaccumulation potential based on study results
Based on the results of this study, we can conclude that MPKO reached the blood when administered by inhalation.

Executive summary:

Blood levels of 2-pentaonone oxime (2-PO; MPKO) and its metabolite methyl propyl ketone (MPK) were measured with inhalation exposure in a GLP study in which five male rats were exposed nose-only to the oxime at an average concentration of 297 ppm. Blood samples for analysis were collected prior to exposure, at 2, 4, and 6 hours during the exposure and 6 hours following termination of the exposure. Analysis was performed using a validated method.

Both detectable levels of 2-PO and MPK were present following 2 hours of exposure (average 10.2 and 1.3 mg/kg blood, respectively) and concentrations increased during the 6-hour exposure period and rapidly declined following exposure such that detectable levels [LOD (limit of detection)  = 2 mg/kg blood] were no longer present 6 hours after the termination of the exposure. These results indicate that 2-pentanone oxime reaches the blood with inhalation producing systemic exposure.

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting 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 412 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

yes

Remarks:

The temperature and relative humidity in the exposure unit or animal room occasionally deviated from target limits. These deviations were considered not to have affected the validity of the study.

GLP compliance:

yes (incl. QA statement)

Remarks:

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

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

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

Type of inhalation exposure:

nose only

Vehicle:

air

Details on inhalation 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 evaporator 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.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The actual concentration of the test material in the test atmospheres was measured by total carbon analysis. The response of the analyser was recorded on a PC every minute using a CAN transmitter. The responses of the analysers were converted to concentrations by means of calibration graphs. For each exposure day, the mean concentration was calculated from the values determined every minute. Representative test atmosphere samples were taken continuously from the exposure unit at the animals’ breathing zone and were passed to the total carbon analyser (TCA) through a sample line.

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.:

289.9 ppm (analytical)

Remarks:

+/- 3.1 ppm

No. of animals per sex per dose:

5

Control animals:

yes

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: daily

BODY WEIGHT:
- Time schedule for examinations: before start of exposure and then twice weekly

FOOD CONSUMPTION:
- Food consumption was measured over two 7-day periods (males) or a 7-day period followed by a 6-day period (females), starting on day 0.
The results were expressed in g per animal per day.

BLOOD SAMPLE COLLECTION
To enable possible future determination of the concentration of test material in blood, two blood samples were collected from male animals of the control group and the high-concentration group. Heparin was used as anticoagulant. The samples were stored frozen (-70 ̊C).

HAEMATOLOGY:
-Time: at the end of the treatment period (one or two days before scheduled sacrifice)
- Animals: on all animals
-Fasting: overnight fasting (water was freely available).
- Anaesthesia: CO2/O2 or pentobarbital anaesthesia
- Anticoagulant: EDTA
-Parameters checked: red blood cells (RBC), haemoglobin (Hb), packed cell volume (PCV) reticulocytes, total white blood cells (WBC) differential white blood cells1 prothrombin time (PT) thrombocytes

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: collected from fasted animals in the same way and at the same time as the samples for haematology
- Anticoagulant: heparin
- Parameters checked: alkaline phosphatase activity (ALP), aspartate aminotransferase activity (ASAT), alanine aminotransferase activity (ALAT), gamma glutamyl transferase activity (GGT), bilirubin (total), total protein, albumin, cholesterol (total), phospholipids, triglycerides, creatinine, urea, inorganic phosphate (PO4), calcium (Ca), ratio albumin to globulin (calculated), glucose, chloride (Cl), potassium (K), sodium (Na)

ORGAN WEIGHTS
- At scheduled necropsy of the animals, as soon as possible after dissection
- Preservation: 10% solution of Formalin in a neutral aqueous phosphate buffer (final formaldehyde concentration 4%)
- Organs: adrenals, brain, heart, kidneys, liver, lung with trachea and larynx, ovaries, spleen, testes, thymus, thyroid, uterus

Sacrifice and pathology:

SACRIFICE
- Animals were sacrificed on day 14, after overnight fasting, by exsanguination from the abdominal aorta. The animals were anaesthetized by intraperitoneal injection of sodium pentobarbital.

HISTOPATHOLOGICAL EXAMINATION:
- embedded in paraffin wax
- sectioned at 5 μm
- stained with haematoxylin and eosin
- animals: low- and mid- concentration groups were not processed, except for the nose which was decalcified and embedded in paraffin concurrently with the nose of the control animals and the high concentration group
- Histopathological examination: light microscopy on all tissues and organs listed above. In addition, all gross lesions were examined in the low- and mid-concentration groups.

Statistics:

Tests were performed as two-sided tests with results taken as significant where the probability of the results is <0.05 or <0.01.
Statistical significance was determined with An(c)ova, Kruskal-Wallis and Dunnett's test.
Details regarding the statistical analysis of the results is presented in the section 'any other information on materials and methods'.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Details on results:

CLINICAL OBSERVATION AND MORTALITY
All animals survived until scheduled sacrifice. No abnormalities were observed in animals exposed to the test material or to clean air. No abnormalities were seen at the group-wise observations made about halfway each 6-hour exposure period.

BODY WEIGHT
All animals gained comparable body weight during the 14-day exposure period. Mean body weights of animals exposed to the test material were similar to the control (clean air) body weights throughout the study.

FOOD CONSUMPTION
Food consumption was not affected by the exposure to the test material.

HAEMATOLOGY
Red blood cell and coagulation values and total and differential white blood cell values showed no treatment-related changes. A few statistically significant differences between animals exposed to the test material and the clean air controls occurred but these findings were not ascribed to treatment, namely:
- Higher mean corpuscular haemoglobin (MCH) in females of the low concentration group. There was no concentration-related response. Moreover, the measured red blood cell parameters (red blood cell count, haemoglobin or packed cell volume) showed no significant changes.
Therefore, the difference in the calculated parameter MCH was considered to be a chance finding.
- Higher number of thrombocytes in males of the mid-concentration group. This difference was not ascribed to treatment because there was no concentration- related response.
- Higher percentage of lymphocytes and lower percentage and absolute number of neutrophils in females of the low- and high-concentration groups. In the absence of a concentration-related response these differences were considered to be chance findings.

CLINICAL CHEMISTRY
Clinical chemistry results showed no treatment-related changes. The few statistical significances observed were considered to be unrelated to treatment because the data showed no concentration-related response (males: higher albumin/globulin ratio in the low- and high-concentration groups, higher calcium in the mid- concentration group; females: lower ASAT in all exposed groups, lower albumin/globulin ratio in the low- and mid-concentration groups).

ORGAN WEIGHTS
The organ weight results showed the following statistically significant differences between animals exposed to the test material and clean air controls:
- Higher spleen weight (absolute and relative to body weight) in males of the mid-and high-concentration groups compared to control group animals (absolute: +13.2% and +17.2%; relative: +17.2% and 17.2%).
- Higher weight of the ovaries (absolute and relative to body weight) in females of the mid-concentration group compared to control group animals. This finding was not ascribed to treatment because there was no concentration-related response.

PATHOLOGY
-Macroscopic examination: There were no macroscopic findings attributable to the exposure to the test material. The few gross changes observed represented background pathology in rats of this strain and age and occurred only incidentally.
-Microscopic examination: Microscopic examination did not reveal treatment-related histopathological changes. The few histopathological changes observed in the high-concentration group were considered unremarkable because they represented background findings and occurred in only one or two animals or at about the same incidence in the high-concentration group and the control group.

Dose descriptor:

NOAEC

Effect level:

149.3 ppm (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Dose descriptor:

NOAEC

Effect level:

616.7 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Remarks on result:

other: Conversion in mg/m3 according to M. J. Derelanko, The Toxicologist's Pocket Handbook, Second Edition, 2008, Table 50 Conversion Table for Gases and Vapours

Critical effects observed:

yes

Lowest effective dose / conc.:

616.7 mg/m³ air

System:

haematopoietic

Organ:

spleen

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Monitoring of exposure conditions:

1. ACTUAL CONCENTRATION:

The overall mean actual concentrations (+/- standard deviation) of the test material in the test atmospheres as measured by total carbon analysis were 52.9 (+/- 2.8), 149.3 (+/- 1.9) and 298.9 (+/- 3.1) ppm for the low-, mid- and high-concentration. These were close to the target concentrations (50, 150 and 300 ppm).

2. TIME TO ATTAIN CHAMBER EQUILIBRATION:

The time to reach 95% of the steady state concentration (T95) was calculated to be about 6 minutes (based on a chamber volume of 37 L and airflow of about 20 L/min).

3. NOMINAL CONCENTRATION AND GENERATION EFFICIENCY:

The mean nominal concentrations (+/- standard deviation) were 52.6 (+/- 2.06), 163.5 (+/- 3.35) and 309.6 (+/- 4.01) ppm for the low-, mid- and high-concentration, indicating generation efficiencies of 100.8%, 91.4% and 96.6%, respectively.

4. AIRFLOW, TEMPERATURE AND RELATIVE HUMIDITY:

The overall mean (± standard deviation) chamber airflows were 20.2 (± 0.19), 23.2 (± 0.27), 20.2 (± 0.18) and 23.5 (± 0.24) L/min for the control, low, mid and high exposure groups respectively. The air temperature in the exposure chambers during exposure was generally within the target range of 20–24°C, minor excursions outside the lower end occurred for the control, low and mid exposure groups. The overall mean temperature was 21.4, 21.6, 21.7 and 21.9°C for chambers of control, low, mid and high exposure groups. The relative humidity during exposure was generally within the target range of 30- 70%, minor excursions outside the lower end occurred for groups control, mid and high. The overall mean relative humidity was 39.3, 40.2, 36.1 and 34.9% in chambers of control, low, mid and high exposure groups. The oxygen concentration, measured on the first exposure day, was 20.2% for exposure chamber control, 20.3% for chamber low, 20.5% for chamber mid and 20.6% for chamber high. The carbon dioxide concentration in the test atmospheres, measured on the first exposure day, was 0.491 vol% for exposure chamber control, 0.418 vol% for chamber low, 0.236 vol% for chamber mid and 0.199 vol% for chamber high

Conclusions:

Under the conditions of this 14-day study exposure to 2-PO by inhalation at concentrations up to 298.9 ppm (actual concentration; 6 hours/day, 5 days/week) was tolerated without obvious signs of toxicity. The only finding were higher spleen weights (absolute and relative to body weight) in males of the mid-and high-concentration groups compared to control group animals (absolute: +13.2% and +17.2%; relative: +17.2% and 17.2%). This increase in absolute and relative spleen weights of male animals, starting at the mid dose group, were regarded as adverse, taken into account the multiple effects in the blood and spleen related to haemolytic anaemia observed in the oral OECD 422 with 2-PO and the classification for STOT RE Cat. 2. Therefore, 149.3 ppm (corresponding to 616.7 mg/m3), was determined to be the No-Observed-Adverse-Effect Concentration (NOAEC) for local and systemic toxicity.

Cross-reference 3

Reason / purpose for cross-reference:

reference to same study

Reference

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

484-470-6

EC Name:

-

Cas Number:

623-40-5

Molecular formula:

C5H11NO

IUPAC Name:

N-pentan-2-ylidenehydroxylamine

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

52.9 ppm (analytical)

Remarks:

+/- 2.8 ppm

Doses / concentrations 2

Dose / conc.:

149.3 ppm (analytical)

Remarks:

+/- 1.9 ppm

Doses / concentrations 3

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

Examinations

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).

Results and discussion

Test results

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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.