Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Recent and well documented study according to OECD and GLP guidelines.

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 403 (Acute Inhalation Toxicity)
Deviations:
yes
Remarks:
The relative humidity in the animal room was outside the target range on several occasions. This deviation is considered not to have affected the validity of the study.
GLP compliance:
yes (incl. certificate)
Test type:
standard acute method
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Methyl propylketoxime (MPKO)
-Chemical name: 2-petanone oxime (2-PO)
-Total quantity: 11kg; one 5 Gallon container
-Batch number: 12060011
-Purity: 99.9%
-CAS Registry Number : 623-40-5
-Hygroscopy : slight
-Storage conditions: ambient temperature
-Approved for use until: 25 October 2015

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: colony maintained under SPF-conditions by Charles River
- Age at study initiation: 9 weeks
- Weight at study initiation: males: 378.6 g; females: 200.2 g
- Fasting period: during exposure the animals had no access to feed or water and were housed individually in the holders.
- Housing: macrolon cages with bedding of wood shavings with environmental enrichment (wooden block and strips of paper)
- Diet (e.g. ad libitum): commercially available 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 exposure. The feed was provided as pellets in the built-in food hopper in the stainless steel wire cage lid.
- Water (e.g. ad libitum): ad libitum from the arrival of the animals until the end of the study, except during exposure. Each cage was supplied with 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.
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
-Temperature (°C): 22 +/- 2°C
- Humidity (%): 55 +/- 10%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): Lighting was artificial by fluorescent tubes, time switch controlled at a sequence of 12 hours light, 12 hours dark (lights on from 7.00 a.m. to 7.00 p.m.).

IN-LIFE dates: From 20 november 2013 until 4 december 2013

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure:
nose only
Vehicle:
air
Remarks:
humidified
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: nose-only exposure units consisting of a cylindrical column, surrounded by a transparent cylinder
- Exposure chamber volume: The column has a volume of 50 - 60 liters.
- Method of holding animals in test chamber: plastic animal holders (Battelle), positioned radially around the central column
- Temperature, humidity, pressure in air chamber: The air entering the unit was controlled at 22 ± 3 ̊C and the relative humidity was maintained between 30 and 70%, if possible. Temperature and relative humidity were recorded about every 30 minutes.

TEST ATMOSPHERE
- Brief description of analytical method used:
The inhalation equipment has been designed to generate a continuous supply of fresh test atmosphere. To generate the test atmosphere, a syringe pump controlled amount of test material was allowed to evaporate in a mass flow controlled stream of humidified air, by directing it through a thermal bath at 65.0 ̊C. The resulting test atmosphere was cooled by leading it through a condenser and subsequently led to the noses of the animals. The animals were placed in the exposure unit after stabilization of the test atmosphere (38 minutes between start of the test atmosphere generation and placement of the animals).
The actual concentration of the test material in the test atmosphere was monitored with a total carbon analyzer (TCA) (Ratfisch RS55T, Munich Germany). Test atmosphere samples were taken continuously from the exposure unit at the animals’ breathing zone and were passed to the TCA.

Analytical verification of test atmosphere concentrations:
yes
Remarks:
The response Y of the analyzer (in % of full scale) was linearly related to the concentration X of the test material (in ppm) with a coefficient of determination of 0.9972: Y = 0.2148 * X – 1.950
Duration of exposure:
4 h
Concentrations:
Actual concentration: 295.0 ppm +/-8.2 ( n=240)
Nominal concentration: 321.7 ppm
No. of animals per sex per dose:
5
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Examinations performed:
1. Clinical signs: On the exposure day, the animals were observed for clinical signs just before exposure, four times during exposure, and twice after exposure. During the observation period, the rats were inspected once daily and, if necessary, handled to detect signs of toxicity. The observations included, but will not be restricted to, the signs listed in Annex 2, except during exposure when animal observation is limited due to the animals’ stay in restraining tubes. During exposure, attention was directed to breathing abnormalities and restlessness. All abnormalities, signs of ill health, and reaction to treatment were recorded. On 1 December 2013, the clinical signs had not been registered in the data collection system. This was corrected retrospectively on 3 December 2013.
2. Body weights: The body weight of each animal were recorded just prior to exposure (day 0), and 1, 3, 7 and 14 days after exposure.
3. Pathology: The animals were sacrificed for necropsy 14 days after exposure by exsanguination from the abdominal aorta under sodium pentobarbital anaesthesia. At necropsy abdominal and thoracic organs were examined in situ.

Results and discussion

Effect levels
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 295 ppm
Based on:
test mat.
Exp. duration:
4 h
Mortality:
No mortality occurred and all animals were sacrificed at the end of the 14-day observation period.
Clinical signs:
other: All ten animals showed a decreased breathing rate during the 4-hour exposure period. Dyspnoea was observed in one female and two male animals during exposure, but was not seen just before the end of the exposure period. Shortly after exposure, these clini
Body weight:
The animals showed body weight loss on day 1, which can be attributed to the restraint during exposure. During the 14-day observation period, all animals showed body weight gain .
Gross pathology:
Macroscopic examination at necropsy revealed abnormalities in the lungs consisting of a gray discoloration (4 males, 1 female) and red spots on one or more lung lobes (2 males, 1 female; Table 4). The grey discoloration of the lungs can be considered a treatment related finding. It cannot be ruled out that the red spots are also a treatment-related finding. There were no macroscopic abnormalities seen in one male and three female animals.

Any other information on results incl. tables

Analytical results

Actual concentration

The actual concentration (+/- standard deviation, number of measurements) in the test atmosphere based on total carbon analysis of the volatile fraction of MKPO was 295.0 ppm (+/-8.2, n=240).

Nominal concentration

The nominal concentration, calculated from the test material flow and the air flow was 321.7 ppm, indicating a generation efficiency of 91.7%. This is slightly lower than expected for vapors and is caused by uptake of the test material by the animals. This can be seen from the nominal concentration prior to placement of the animals in the exposure chamber, which was 303.2 ppm with an actual concentration of 300.1 ppm, indicating a generation efficiency of 99.0%.

Temperature, relative humidity, oxygen concentration and flow

The average temperature in the exposure chamber (± SD, range) was 22.9 ̊C0.1, 22.823.1). The average relative humidity in the exposure chamber (± SD, range) was 46.6% (± 1.4, 45.349.2). The oxygen concentration, as measured in the exposure unit during exposure, was 19.9%. The total air flow was 12.1 Ln/min2 (13.2 L/min).

Applicant's summary and conclusion

Conclusions:
Since none of the animals died during the study, the 4-hour LC50 of MKPO in rats is greater than 295 ppm.
Executive summary:

An acute inhalation toxicity study was performed, according to the OECD guideline 403 and GLP compliance. The aim of the present study was to determine the acute inhalation toxicity of MKPO by exposing five male and five female rats to a target limit concentration of 300 ppm for four hours. Thereafter, the animals were kept for an observation period of 14 days. To detect adverse effects, clinical observations were recorded during and after exposure, body weight was measured before exposure (day 0) and on days 1, 3, 7 and 14, and animals were examined for gross pathological changes at the end of the observation period.

The mean actual concentration (± standard deviation) during exposure was 295.0 (± 8.2) ppm.

All animals showed a decreased breathing rate during the 4-hour exposure period. Dyspnoea was observed in one female and two male animals during exposure, but was not seen just before the end of the exposure period. Shortly after exposure, these clinical abnormalities were no longer observed. Clinical abnormalities observed following the exposure were limited to shallow breathing in two male animals between days 0-3 or days 0-1, respectively. No treatment-related effects on body weight were seen. No mortality occurred and all animals were sacrificed at the end of the 14-day observation period.

Macroscopic examination of the lungs at necropsy revealed a gray discoloration of the lungs (4 males, 1 female) and red spots on one or more lung lobes (2 males, 1 female). No macroscopic abnormalities were seen in one male and three female animals.

Since none of the animals died during the study, the 4-hour LC50 of MKPO in rats is greater than 295 ppm.