Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Cross-reference
Reason / purpose for cross-reference:
data waiving: supporting information
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Principles of method if other than guideline:
This study was designed to further investigate the effect of MEKO on the olfactory epithelium and establish a NOAEC, determine if reversible with recovery phase. The mouse was chosen since it appears to be more sensitive to this effect than the rat. Liver peroxisome proliferation and glutathione content were also evaluated.
GLP compliance:
yes
Limit test:
no
Species:
mouse
Strain:
CD-1
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Char1es River Laboratories, Portage, Michigan
- Age at study initiation: 7 weeks
- Weight at study initiation: 27.6 (25.0-30.0) g
- Fasting period before study: no
- Housing: Animals were doubly housed in suspended, stainless steel, wire mesh cages during the first week of the acclimation period and individually housed during the remainder of the acclimation period and all other non-exposure periods. During exposures, animals were individually housed in
wire mesh, stainless steel cages.
within a 6000 l i t e r glass and
stainless steel exposure chamber
- Diet (e.g. ad libitum): Certified mash 1aboratory diet (Purina Mills Rodent Diet #5002). Fresh food presented as required.
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 (average)
- Humidity (%): 46 (average)
- Photoperiod (hrs dark / hrs light): 12/12


Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
air
Details on inhalation exposure:
Chamber environmental conditions averaged 21°C and 49% relative humidity. Mass and particle size distribution measurements during chamber trials indicated that there was no measurable amount of MEKO likely to be present as an aerosol.

Pre-Study Tria1s : Trial s were performed to evaluate the optimal set o f equipment and operating conditions to generate a stable atmosphere at the targeted exposure levels.

Chamber Operation : The exposure chambers were operated dynamically under slight positive pressure. The chamber's airflow rate, total flow rate, time for air change and 99% equilibrium time (T99) for each group were determined. The chamber size and airflow rate was considered adequate to maintain the animal loading factor below 5% and oxygen above 19%. The chamber was exhausted through a system consisting of a coarse filter , a
HEPA filter and into an incinerator. Recordings o f chamber temperature, relative humidi ty, airflow rate and static pressure were made every ha1 f-hour during exposure. The volatilization chamber consisted of two concentric glass tubes sealed at the ends. The outer tube had ports for the delivery of MEKO and nitrogen and the exhaust of the vapor-laden atmospheres. The inner glass tube was equipped with a glass coil spiraling down around the outside of the tube and an internal heating element. MEKO was pumped onto the top of the glass coil, where it flowed down and around the heted tube. Heated nitrogen was introduced a the bottom of the volatilization unit to flow upward, counter to the flow of MEKO, to maximize volatilization. The vapors were generated under nitrogen to get the vapor-laden atmospheres near or below the lower-explosion-limit prior to mixing with air. The output of the volatilization chamber was connected to the entry turret of the exposure chamber, where it mixed with air prior to entrance into the animal compartment of the chamber. The temperature of the volatilization unit was measured using an alarmed monitoring system.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Determination of the MEKO exptsure level s were made using a MIRAN Ambient Air analyzer equipped with a strip chart recorder. The test atmospheres were drawn at one hour intervals through the MIRAN and measurements were recorded at least hourly during the exposures from the normal sampling portal and once during exposures from the distribution sampling portal. The exposure levels were determined by comparison of the measured absorbance to a calibrated response curve constructed using the same instrument settings.

Particle size distribution measurements were performed once during each trial for the chambers and room ai r using a TSI Aerodynamic Particle Sizer equipped with a diluter. The samples were drawn for 20 seconds at a rate of 5 liters per minute. The mass median aerodynamic diameter, geometric standard deviation and percent of particles 11.0, 54.0 and 510 microns were calculated. A computer was used to program the system to the appropriate settings prior to sampling. The particle size distribution s were calculated by the computer and printed out following an analysis of an assumed lognormal distribution .
Duration of treatment / exposure:
Main study: 1, 2, 4 or 13 weeks.
Satellite groups: additional recovery period of 4 or 13 weeks
Frequency of treatment:
6 h/day, 5 days/week
Dose / conc.:
3 ppm (analytical)
Remarks:
± 0.1 ppm (10.8 mg/m³)
Dose / conc.:
10 ppm (analytical)
Remarks:
± 0.3 ppm (36 mg/m³)
Dose / conc.:
30 ppm (analytical)
Remarks:
± 1.0 ppm (108 mg/m³)
Dose / conc.:
100 ppm (analytical)
Remarks:
± 2.0 ppm (360 mg/m³)
No. of animals per sex per dose:
Main study: 10 males/dose/interval (exposures for 1, 2, 4 or 13 weeks)
Satellite groups: 5 males/dose/interval (satellite animals were removed after 1, 2, 4 or 13 weeks and allowed to recover for 4 or 13 weeks)
Control animals:
yes
Details on study design:
- Dose selection rationale: Exposure levels were selected by the Sponsor on the basis of available toxicity data.
- Rationale for selecting satellite groups: In a previous inhalation oncogenicity study of MEKO in mice and rats (in which exposures to MEKO were at concentrations of 15, 75 and 374 ppm for 6 hours/day, 5 days/week for 2 years), MEKO was found to be a liver oncogen in male mice at 374 ppm and male rats at 75 ppm. In addition, olfactory degeneration was seen in both rats and mice in all MEKO exposure groups. For this reason, the current study was designed to investigate the time course of development and reversibility of the degeneration seen i n the olfactory region of the nasal turbinates. Only male mice were used in this study because there was no gender difference in the response and the mice seemed. to be more sensitive than the rats to this effect.
- The 3 and 10 ppm MEKO exposures were chosen because 15 ppm was not a no observed effect level in the previous chronic study. The higher exposure levels of 30 and 100 ppm, along with the 1-,2-, 4- or 13 week exposure durations and the 4 and 13 week recovery after each exposure duration were selected in order to evaluate the time course for development of the olfactory epithelial lesion and its reversibility.
Observations and examinations performed and frequency:
Mortality and Gross Signs of Toxicologic or Pharmacologic Effects: Twice daily.

For Abnormal Signs: Daily. Animals were observed as a group once during each exposure.

Detailed Physical Examinations: Pretest and weekly thereafter.

Body Weight: Twice pretest, weekly during study period and prior to scheduled sacrifice.

Sacrifice and pathology:
Post Mortem Examination: Microscopic examination was performed on the nasoturbinates of all animals. Degenerative changes in each individual section examined were scored for severity for a relative comparison among the exposure groups. Severity scores were based on the subjective assessments of degree of degeneration (decrease in thickness) and the approximate percentage of the olfactory epithelium affected. The severity scores defining the approximate extent of tissue involvement were: Minimal, up to 2%; Slight, from 2 to 10%; Moderate, from 10 to 30%; Moderately Severe, from 30 to 70%; and Severe, over 70%.

Generalized maps were prepared to show the distribution of morphological changes in the olfactory epithelium. A comprehensive set of diagrams suitable fo recording the location and distribution of tissue responses for each transverse sectional level of the nose was used for the mapping procedure.

Peroxisome Proliferation: For the animals sacrificed after 13 weeks of exposure, potential peroxisome proliferation in the liver was evaluated using electron microscopy o f selected liver samples and by examining the effect of MEKO on mouse hepatic peroxisomal fatty P-oxidation.
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):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
At the end of the 1, 2, 4 and 13 week exposure periods, degeneraton of the olfactory epithelium lining the dorsal meatus was seen in the anerior region of the nasal cavity. In a few instances, the olfactory epithelium covering the tips of the nasoturbinal scrolls projecting into the dorsal region of the nasal cavity was also degenerated. Large areas of olfactory epithelium lying laterally and posteriorly were unaffected. In general, approximately 10% or less of the total olfactory tissue was affected. In several instances, the degenerated olfactory epithelium was reepithelialized by squamous/squamoid and /or respiratory types of epithelium. Degeneration, which was dose-related in incidence and severity, was seen in mice exposed to 30 and 100 ppm after 1 week of exposure and in several mice exposed to 10 ppm after 13 weeks of exposure. The incidence and severity of the degeneration present after 1 week of exposure did not increase with the longer exposures. The olfactory degeneration was reversible. Recovery was complete within 4 weeks following exposures at 10 ppm and nearly complete withn 13 weeks after exposures at 30 and 100 ppm.
Histopathological findings: neoplastic:
no effects observed
Details on results:
The 3 ppm concentration was considered the NOAEC.



Dose descriptor:
NOAEC
Effect level:
10.8 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: olfactory epithelium degeneration in the nasal cavity
Critical effects observed:
yes
Lowest effective dose / conc.:
36 mg/m³ air (analytical)
System:
respiratory system: upper respiratory tract
Organ:
nasal cavity
Treatment related:
yes
Dose response relationship:
yes
Conclusions:
Under the conditions of this study, inhalation exposure to MEKO at concentrations of 10, 30 or 100 ppm for 6 hours/day, 5 day/week for 13 weeks produced minimal to moderately severe olfactory epithelium degeneration in male CD-1 mice. The incidence and severity of the degeneration was concentration dependent and not progressive over time with continued exposure. The lesions were localized to the olfactory epithelium lining the dorsal meatus in the anterior portion of the nasal cavity. Large areas of olfactory epithelium laterally and posteriorly appeared unaffected. No effects to olfactory epiithelium were found at 30 ppm MEKO in > 50% of the animals, indicating that only the most sensitive animals responded at this relatively high level of exposure. The degeneration of the olfactory epithelium following exposure to MEKO is not widespread but limited primarily to cells in the dorsal anterior portion of the nasal cavity. This represents approximately 10% of the olfactory epithelium lining the nasal cavity. The effect was reversible with cessation of exposure with complete recovery observed within 4 weeks at 10 ppm and nearly complete recovery after 13 weeks at the higher concentrations. The low incidence of lesions that occurred at 10 ppm only after 13 weeks of exposure suggests that this exposure concentration is near the threshold for the olfactory effects of MEKO. The NOAEC was considered to be 3 ppm.
Under the exposure conditions of this study, MEKO did not induce hepatic peroxisome proliferation nor did it cause any ultrastructural changes in liver cells after 13 weeks of exposure at concentrations up to 100 ppm. However, significant increases in levels of hepatic non-protein sulphydryl groups (primarily reduced glutathione) were measured following MEKO exposures at 30 and 100 ppm.

Data source

Materials and methods

Results and discussion

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion