Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 27 May 2008 to 20 Feb 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed according to international test guidelines and to GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.2 (Acute Toxicity (Inhalation))
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.1300 (Acute inhalation toxicity)
Deviations:
no
GLP compliance:
yes
Test type:
standard acute method
Limit test:
yes

Test material

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

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories Ltd, Laboratory Animals Services, Wölferstrasse 4, 4414 Füllinsdorf, Switzerland
- Age at study initiation: 9 weeks for males and 10 weeks for females
- Weight at study initiation: 241.9 to 264.1 g for males and 197.1 to 214.7g for females
- Fasting period before study: no
- Housing:In groups of five in Makrolon type-4 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ Schill AG, 4132 Muttenz / Switzerland).
- Diet (e.g. ad libitum):Pelleted standard Kliba Nafag 3433 (batch no. 12/08) rat maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum except during the period when the animals were restrained in exposure tubes. The feed batch was analyzed for contaminants.
- Water (e.g. ad libitum):Tap-water was available ad libitum in water bottles except during the period when the animals were restrained in exposure tubes. Results of bacteriological assay, chemical and contaminant analyses of representative samples are archived at Harlan Laboratories Ltd.
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light):12/12

IN-LIFE DATES: From 20 Aug 2008 to 03 Sep 2008

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus:Inhalation exposure was performed using a system similar to that originally described by Sachsse et al..
- Method of holding animals in test chamber: The animals were confined separately in restrained tubes which were positioned radially around the flow-past, nose-only exposure chamber as described by Cannon et al.. The design of this chamber is based upon the fluid dynamic modeling of the test aerosol flow.
- Source and rate of air: The exposure system ensured a uniform distribution and provided a constant flow of test material to each exposure tube. The flow of air at each tube was 1 L/min, which was sufficient to minimize re-breathing of the test atmosphere as it was more than twice the respiratory minute volume of a rat.
- System of generating particulates/aerosols: The test aerosol was generated using a plastic nebuliser connected to a syringe pump. The
polyethylene injector inside model was replaced by a stainless steel injector.
- Method of particle size determination: The cumulative particle size distribution of the test atmosphere was determined using a 7-stage Mercer cascade impactor. The test atmosphere was impacted at each stage onto an appropriate medium and the particle size distribution of the test item in the generated atmosphere was measured by gravimetrically analyzing the test item deposited on each stage of the cascade impactor.
- Temperature, humidity, pressure in air chamber: The relative humidity and temperature in the chamber were measured continuously during each
exposure using a calibrated device. The results are reported at 30 minute intervals from the start of exposure during the whole treatment.

TEST ATMOSPHERE
- Brief description of analytical method used:Test atmosphere samples were collected on Millipore® durapore filters, Type HVLP, (Polyvinylidenedifluoride membrane, pore size 0.45 μm) loaded in a 47 mm in-line stainless steel filter sampling device. The duration of sampling was sufficient to ensure reliable results. The weighing of these filters was delayed for 30 seconds to 3 minutes after sampling, in order to attain stable filter weights
- Samples taken from breathing zone: yes

TEST ATMOSPHERE: see details in the table below.
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Concentrations:
5.6 mg / L air
No. of animals per sex per dose:
- 5 animals/sex in the control group
- 10 animals/sex in the treatment group
Control animals:
yes
Details on study design:
- Duration of observation period following administration: 1 or 15 days
- Frequency of observations: Once before exposure on the day of exposure (test day 1), once per hour during exposure, once after exposure on test day 1 and twice daily during the observation period.
- Frequency of weighing:
- Main study animals (sacrificed after a 15-day observation period): On test days 1 (before exposure), 4, 8 and 15 (before necropsy)
- Interim sacrifice animals (sacrificed after a 1-day observation period): On test days 1 (before exposure) and 2 (before necropsy).
- Necropsy of survivors performed: yes
- Other examinations performed:
- clinical signs: Once per hour during exposure (only grossly abnormal signs as animals were restrained), once
after exposure on test day 1 and once daily during the observation period.
- histopathology:The nasal cavities of all animals (except of one animal of group 1) were processed, embedded in paraffin, cut at a nominal thickness of 2-4 micrometers, stained with hematoxylin & eosin (H&E) and examined by light microscope.

Results and discussion

Effect levels
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 5.6 mg/L air
Based on:
test mat.
Exp. duration:
4 h
Mortality:
No mortality occured during the study.
Clinical signs:
other: Slight to moderate salivation was recorded in all animals given the test item during exposure only. Ruffled fur was noted in all animals on test day 1 after the end of exposure, lasting up to test day 3. From test day 4 onwards, the animals were free from
Body weight:
No treatment-related effects on body weight were noted in animals given the test item.
Gross pathology:
No macroscopical findings were noted at scheduled necropsy.
Other findings:
- Histopathology:
Different degrees of necrosis of the olfactory epithelium were recorded one day after exposure in the nasal cavities of males and females receiving the test item, mainly at levels II-IV. These findings were sometimes noted along with luminal hemorrhages. After an observation period of 15 days, there was no necrosis at any level and signs of degeneration/regeneration of the olfactory epithelium and findings were limited to levels II and III.
No findings were noted in the nasal cavities of control animals.

Applicant's summary and conclusion

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test material is not harmful by the inhalation route.
Executive summary:

In a acute inhalation toxicity study (Harlan Laboratories Ltd 2009 study no. B90527) one group of 10 male and 10 female Wistar rats were exposed by nose-only, flow past inhalation, for a single 4-hour period, to an aqueus dilution of Dimethyl 2-methylglutarate at a chemically determined mean aerosol concentration of 5.6 mg/L air. Half of the animals was necropsied after a 1-day observation period, the other half was necropsied after a 15-day observation period. A further group of 5 male and 5 female rats served as air control (exposed to air only) and were sacrificed after a 15-day observation period.

Acute inhalation LC50> 5.6 mg/L air.

 

Dimethyl 2-methylglutarate is not classified based on the LC50 in rats.

 

No mortality was observed. The clinical signs consisted of slight to moderate salivation recorded in all animals given the test item during exposure only. Ruffled fur was noted in all animals on test day 1 after the end of exposure, lasting up to test day 3. From test day 4 onwards, the animals were free from clinical signs.

There was no treatment-related effect on body weight.

 Histopathological examination showed different degrees of necrosis of the olfactory epithelium which were recorded one day after exposure in the nasal cavities of males and females receiving the test item, mainly at levels II-IV. These findings were sometimes noted along with luminal hemorrhages. After an observation period of 15 days, there was no necrosis at any level and signs of degeneration/regeneration of the olfactory epithelium and findings were limited to levels II and III.

 

This acute inhalation study is classified as acceptable. It does satisfy the guideline requirement for an acute inhalation toxicity study (OECD 403) in the rat.