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Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is recently performed according to OECD 412 and GLP, and well documented.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
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. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Chemical name: 2-pentanone oxime (2-PO)
CAS reg number: 623-40-5
Batch number: 12060011
Purity: 99.9%
Appearance: clear and colourless liquid
Molecular formula: C5H11NO
Molecular weight: 101.15 g/mol
Date of receipt: 28 October 2013
Total quantity received: 11 kg; one 5 gallon container
Approved for use until: 25 October 2015
Storage conditions: ambient temperature
Supplier: Sponsor
TNO Triskelion Dispense Reference number: 13021E

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals 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.

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure:
nose only
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
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
298.9 +/- 3.1 ppm
Basis:
other: actual concentration
Remarks:
Doses / Concentrations:
149.3 +/- 1.9 ppm
Basis:
other: actual concentration
Remarks:
Doses / Concentrations:
52.9 +/- 2.8 ppm
Basis:
other: actual concentration
No. of animals per sex per dose:
5
Control animals:
yes

Examinations

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'.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
no treatment-related changes
Food consumption and compound intake (if feeding study):
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
no treatment-related changes
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
no treatment-related changes
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
non-adverse treatment-related effect.
Gross pathological findings:
no effects observed
Histopathological findings: neoplastic:
no effects observed
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.
- 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.

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEC
Effect level:
298.9 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse and treatment-related effects were observed up to and including the highest tested dose level
Key result
Dose descriptor:
NOAEC
Effect level:
1 254 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse and treatment-related effects were observed up to and including the highest tested dose level

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

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 2024°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

Applicant's summary and conclusion

Conclusions:
Under the conditions of this 14-day study exposure to MPKO by inhalation at concentrations up to 298.9 ppm (actual concentration; 6 hours/day, 5 days/week) was tolerated without obvious signs of toxicity. Therefore, the highest concentration tested in this study, 298.9 ppm (corresponding to 1254 mg/m3 at 22°C and 1013 hPa), was a No-Observed-Adverse-Effect Concentration (NOAEC) for local and systemic toxicity.
Executive summary:

A subacute inhalation toxicity study was performed, according to the OED guideline 412 and GLP compliance. The aim of this study was to examine the sub-acute (14-day) inhalation toxicity of the test material MPKO in rats. To this end, four main groups of male and female Sprague Dawley rats (5/sex/group) were exposed nose-only to concentrations (overall mean ± standard deviation) of 52.9 (± 2.8), 149.3 (± 1.9) and 298.9 (± 3.1) ppm for 6 hours/day, 5 days/week, during 2 consecutive weeks. The highest target concentration was selected just below the maximum satured vapour concentration. The study included a concurrent control group of the same size which was exposed to humidified air only.

The clinical pathology and organ weight results showed a few statistically significant differences between a group of exposed rats and the clean air control group. However, most of these differences were chance findings unrelated to treatment because the data showed no concentration-related response.

The weight of the spleen (absolute and relative to body weight) was higher in males of the mid- and high-concentration groups than in controls. Considering the effects on spleen weight previously found in oral studies with MPKO (Honeywell Reports MA-RR-12-4351 and MA-RR-12-4352), this finding is considered to be treatment- related. However, since the magnitude of difference is relatively small (16% for the relative weight), and no corroborative changes occurred e.g. in haematology parameters or spleen morphology, this effect on spleen weight in male animals is not considered adverse.

Exposure to the test material up to 298.9 ppm (mean actual concentration measured by total carbon analysis) was well tolerated as evidenced by the absence of clinical signs of toxicity or adverse effects on growth, food consumption, routine haematology and clinical chemistry values, organ weights and pathology findings.

Therefore, the highest concentration tested in this study, 298.9 ppm (corresponding to 1254 mg/m3 at 22°C and 1013 hPa) was a No-Observed-Adverse-Effect Concentration (NOAEC) for local and systemic toxicity. The target concentration of 300 ppm (= actual concentration of 298.9 ppm) was set just below the maximum saturated vapor concentration for MPKO.

Based on the measurement of MPKO and its metabolites in the blood of the rats after a single (6 -hour) exposure by inhalation (Report #V20451/01) it is concluded that MPKO has reached the blood, and therefore the systemic exposure has occured at the highest attainable exposure condition.