Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 - 25 July, 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Version / remarks:
Some rats have been sacrified with carbon dioxide inhalation instead of carbon dioxide/oxygen inhalation which is not considered to have affected the integrity of the study because insignificant difference between the 2 anesthesics.
3 animals inadvertently had their organs weighed at the time of sacrifice.Not considered have affected the integrity of the study because more data than necessary was recorded and these values were not used in the statistical evaluation of mean organ weights.
1 animal inadvertently had a macroscopic skin lesion. This was not required tissue. Not considered have affected the integrity of the study because more data
than required was recorded and approved by the Sponsor.
Deviations:
not applicable
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
Supplier : Thiokol Propulsion, 9160 North Hwy 83 Bldg M3, Corine, Utah 84307
Lot : 99-99-01 (trials), 114054 (trials), JH-1744-88 (exposures)
- Expiration date of the lot/batch: Not available
- Purity test date: >99%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Kept tightly closed; stored in a cool dry place

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Albino rats Vaf/Plus
Sprague-Dawley-derived (CD)
Crl:CD (SD) IGS BR

The rat is an animal model commonly utilized in toxicity studies. In addition, a historical database is available for comparative evaluation.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Kingston, New York, 12484
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 6 weeks
- Weight at study initiation:
Males = mean 187; Female = mean 149; Individual weights of animals were within +/-20% of the mean weight
- Fasting period before study: No
- Housing:
Housing during non-exposure period : in elevated, stainless steel, wire mesh cages, doubly-housed at receipt and during the first week of the acclimation period and all other non-exposure period
Housing during exposure period : Individually-housed in polycarbonate nose-only tubes attached to a cast aluminium and alloy 40 liter exposure chamber
- Diet and Water ad libitum during non-exposure period and not provided during exposure
- Acclimation period: 2 weeks from reception and 2 days prior to the first exposure and the day preceding the first exposure to the test substance, animals were acclimated to the nose-only cones and chambers for at least 2 hours and for at least 4 hours, respectively.

DETAILS OF FOOD AND WATER QUALITY:
Analytical certification of each feed lot used during the study was performed by the manufacturer.
Water analysis are conduted by Elizabethtown Water Company, to ensure that water meets standards specified under the EPA Federal Safe Drinking Water Act Regulations. In addition, water samples are collected biannually from representative rooms in the Testing facility; chemical and microbiological water analyses are conduted on these samples by a subcontratct laboratory.
No known contaminants in the feed or water were expected to have interfered with the results of this study.

ENVIRONMENTAL CONDITIONS
During non-exposure period, desired temperature = 18-26°C, Actual = 16-26°C
During exposure periods; desired temperature = 20-24°C, Actual = 16-30°C
During non-exposure period, desired Relative Humidity = 30-70%, Actual = 50-78%
During exposure periods; desired Relative Humidity = 40-60%, Actual = 10-61%

- Photoperiod (hrs dark / hrs light): 12-hour light/dark

Administration / exposure

Route of administration:
inhalation: mixture of vapour and aerosol / mist
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
>= 1.6 - <= 2.8 µm
Geometric standard deviation (GSD):
1.8
Remarks on MMAD:
These results indicated that the test substance atmospheres were respirable in size to rats.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Tes test substance was mixed in an appropirately sized Erlenmeyer flask on a stirplate with a magnetic stir br with the vehicle (acetone).
The test substance mixture or the vehicle was fed directly via 1/8'' Teflon tubing from the flask, through a FMI Fluid Metering Pump with a 1/8'' piston and into an air atomizing nozzle. as a vapor/aerosol. House-line air was delivered from a regulator and backpressure gauge, via 1/4'' tubing and connected to a plastic Y tube taht split the airflow into the generation and dilution systems. The generation air (20Lpm) was directed, via 1/4" tubing, through a flowmeter regaulated by a metering valve, and a blackpressure gauge, to the inlet of the air atomizing nozzle to produce the aerosol.

- Temperature, humidity, pressure in air chamber:
During exposure periods; desired temperature = 20-24°C, Actual = 16-30°C
During exposure periods; desired Relative Humidity = 40-60%, Actual = 10-61%

- Method of particle size determination: TSI Aerodynamic Particle Sizer
- Treatment of exhaust air: The chamber were exhausted through a system consisting of a coarse filter, a HEPA filter and an activated charcoal bed.

TEST ATMOSPHERE
- Brief description of analytical method used: Gravimetric analysis of the airborne aerosol concentration for each test substance exposure level and HPLC analysis

VEHICLE: Acetone
- Lot/batch no. of vehicle (if required): 2079, T06253 and N23293
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Check table Doses/Concentrations below.
Exposure levels were determined analytically 4 times per exposure.

The analytically measured levels of airbone test substance were reasonably close to the target exposure levels.
These exposures were essentially an aerosol exposure with a minor vapor component.
Duration of treatment / exposure:
2 weeks
Frequency of treatment:
6 hours per day, 5 day per week, total of 11 exposures
Doses / concentrationsopen allclose all
Dose / conc.:
150 mg/m³ air
Remarks:
Target concentration
Dose / conc.:
165 mg/m³ air (analytical)
Dose / conc.:
500 mg/m³ air
Remarks:
Target concentration
Dose / conc.:
545 mg/m³ air (analytical)
Dose / conc.:
1 500 mg/m³ air
Remarks:
Target concentration
Dose / conc.:
1 313 mg/m³ air (analytical)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Exposure levels were Sponsor specified based on the results of an Acute Inhalation Study. The high exposure level was intented to exceed the limit test of 1000 mg/m3 of the US EPA in OPPTS 870.3465 for subchronic inhalation toxicity study
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: NA
Positive control:
A group of rats received Acetone only.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS and VIABILITY: Yes
- Time schedule: Once in the morning and once in the afternoon
Animals that were in poor health or in a possible moribund condition were identified for further monitoring and possible euthanasia.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once during each exposure
Observations for signs of toxic or pharmacological effects.
- Cage side observations checked in table were included in the attached report of the whole study.

BODY WEIGHT: Yes
- Time schedule for examinations: Twice pretest, weekly during the study and fasted jsut prior necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY AND COAGULATION : Yes
- Anaesthetic used for blood collection: A lightly anesthesized (CO2/O2) via puncture of the orbital sinus (retrobulbar).
- Animals fasted: Yes overnight
- How many animals: All animals surviving to terminal necropsy
- Parameters checked in table in that attached study report were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: A lightly anesthesized (CO2/O2) via venipuncture of the dorsal aorta
- Animals fasted: Yes overnight
- How many animals: All animals surviving to terminal necropsy
- Parameters checked in table in the attached study report were examined.

URINALYSIS: Yes
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes, overnight, 16 hours
- Animals fasted: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: twice pretest and once weekly
- Dose groups that were examined: All animals
- Battery of functions tested: general condition, skin and fur, eyes, nose, oral activity, abdomen and external genitalia, occurence of secretions and excretions and autonomic activity; changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypy or bizarre bahavior.

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
A complete macroscopic postmortem examination was performed on all animals, including animals euthanized prior to study termination of found dead.
Animals were fasted prior to the scheduled sacrifice.

HISTOPATHOLOGY: Yes
Animals showing signs of severe debility, particularly if death appeared imminent, were euthanized to prevent loss of tissue through autolysis.
Terminal necropsies were performed on 5 animals/sex for Groups 1 and 2 and 1 animal/sex for Group 3.

TISSUE/MICROSCOPIC EVALUATION : Yes for all animals in Group 1 and 2 and for all animals in Group 3 that survived until terminal sacrifice. Slides of tissues indicated in the table in the attached study report were prepared and examinated.

METHOD OF EUTHANASIA : Animals were exsanguinated following carbon dioxide/oxygen inhalation except animals euthanized on 13 July 2000 were exsanguinated carbon dioxide inhalation.
Other examinations:
ORGAN WEIGHTS
Organs indicated in the table in the attached study report were taken from all survivors at the scheduled necropsy, weighted, recorded and organ/body and organ/brain weight ratios calculated.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The only-treatment related responses during the exposures were labored breathing and decreased activity noted in the 500 mg/m3 exposure group beginning with the third exposure.

Signs of toxicity noted during the morning or afternoon viability checks during the 2-week exposure period included decreased fecal volume, decreased feed consumption, prostration, yellow stains on the ventral surface, red nasal discharge, irregular gait, lethargy, head bobbing, poor conditions, pale, blackwards walking and labored breathing. These signs were seen most frequently in the 500 and 1500 mg/m3 exposed animals preceding the animals being euthanized or found dead.
Yellow stains on the ventral surface were also frequently noted in the 150 mg/m3 exposed animals.
Irregular gait was also occasionally noted during the afternoon viability checks in the acetone contro and 150 mg/m3 exposed animals; this was probably a vehicle (cns depression) effect.
Clinical signs seen in the 150 mg/m3 group were considered to be of no toxicological importance.
Mortality:
mortality observed, treatment-related
Description (incidence):
There were 18 unscheduled deaths during the study.
1 of the 1500 mg/m3 exposed female was found dead on the morning after the second exposure and ALL of the remaining animals of this group were sacrified in poor health on that day thus terminating this exposure level early.
4 of 5 males and 4 of 5 females exposed to 500 mg/mg3 were euthanized in a moribund conditions or found dead during the 2 weeks of exposure.
2 animals (1 by sex) from this test group survived to study termination.
All animals in the control and 150 mg/m3 groups survived to study termination


Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically meaningful differences in BW in the 150 mg/m3 exposed animals compared to the Acetone control animals.
The 500 mg/m3 exposed animals showed significant less weight gain than the Acetone control animals during the first week of exposure.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A significant decrease in feed consumption (grams/animals/day and grams/kg/day) was noted in the 500 mg/m3 exposed female animals during the first week of exposure.
A statistically significant increase in feed consumption was noted in the 150 mg/m3 exposed male animals during the second week of exposures. However an increase in feed consumption is not considered of toxicological significance
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
There were statistically significant decreases in 150 mg/m3 exposed females for hemoglobin concentration, mean copuscular volume and mean corpuscular hemoglobin and a statistically significant increase un absolute monocytes. HOWEVER, the absolute differences were minimal in the males and were not seen as statistically significant in the males.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
A possible toxicologically meaningful decrease (statistically significant in males) in blood urea nitrogen was noted in the 150 mg/m3 exposed animals.
Urinalysis findings:
no effects observed
Description (incidence and severity):
A possible toxicologically meaningful difference unrine color was noted in the 150 mg/m3 exposed animals. Most of these animals were noteed with yellow urine compared to Acetone control animals which were mostly noted with straw colord urine.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Signs of toxicity included irregular gait, decreased activity, lethargy, head bobbing and labored breathing.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were statistically significant increases in absolute kidney weight for the 150 mg/m3 exposed males and for absolute kidney weights for the V=150 mg/m3 exposed females. HOWEVER, the absolute differences were minimal, were not seen as statistically significant in the other sex and there were no microscopic findings associated.
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
The only test-related microscopic finding was in the LARYNX at the level of the ventral seromucous glands.
This finding considered to be a non-specific direct local effect of the test substance.
Other findings in the larynx and in the other tissues and organs occurred with comparable incidence and severity in the Acetone and 150 mg/m3 groups or they occurred sporadically..
These incidental findings have been seen in the rats of this strain and age used in similar studies conducted in this facility. Squamous metaplasia is a common finding in the rat larynx since the area is very sensitive to insult and is usually an adaptive response and reversible
Histopathological findings: neoplastic:
no effects observed

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 165 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
500 mg/m³ air
System:
other: Clinical signs and mortality
Organ:
other: clinical signs and mortality
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

NA

Applicant's summary and conclusion

Conclusions:
165 mg/m3 was determined to be the NOAEL (analytically determined concentration, whereas the target concentration was 150 mg/m3)