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Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented study report similar or equivalent to OECD 413. GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report date:
2005

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
Baseline gasoline vapor condensate
IUPAC Name:
Baseline gasoline vapor condensate
Details on test material:
- Name of test material (as cited in study report): Baseline gasoline vapor condensate
- Analytical purity: 100%
- Lot/batch No.: API 99-01
- Expiration date of the lot/batch: N/a, stable per MSDS
- Storage condition of test material: Room temperature

Baseline gasoline vapor condensate (API 99-01) is a 20% light fraction (C5-C6) of a whole unleaded gasoline sample. The test material was a representative evaporative emission tested under the US EPA 211(b) Fuels and Fuel Additives Health Effects Testing Program (U.S. Environmental Protection Agency 1994. Fuels and Fuel Additives Registration Regulations; Final Rule pursuant to the Clean Air Act 211(b), 211(e). 40 CFR Part 79, FR59, No. 122: 33042-33142 (27 June 1994). Washington, DC: EPA, 1994).

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Kingston, NY 12484
- Age at study initiation: ~ 4-6 weeks
- Weight at study initiation: Male: 294.5 g; Female: 201.5 g
- Housing: Individually in stainless steel wire mesh cages
- Diet (e.g. ad libitum): Available without restriction
- Water (e.g. ad libitum): Available without restriction
- Acclimation period: at least 16 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15-26
- Humidity (%): 12-79
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 1 m^3 glass and stainless steel exposure chamber
- Method of conditioning air: Houseline nitrogen was delivered from a regulator with a backpressure gauge through a stainless steel fitting to create three flow systems: the test substance pressurization flow, the purge flow and the volatilization flow. As the test substance laden nitrogen was drawn into each of the chambers, it was mixed with room air.
- Air flow rate: 200 L/min
- Air change rate: 12 air changes/hour
- Treatment of exhaust air: The chambers were exhausted through the in house filtering system, which consisted of a coarse filter, a HEPA filter, activated charcoal and then through a fume incinerator.

TEST ATMOSPHERE
- Brief description of analytical method used: During each exposure, measurements of airborne concentrations were performed in the animals' breathing zone at least 4 times using an appropriate sampling procedure and infrared spectrophotometric analytical procedure. Also, one charcoal tube sample was collected per chamber per week and analyzed by GC to characterize at least 10 major components to show test substance stability and comparison between the neat liquid test substance and the vaporized test atmospheres.
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Infrared spectrophotometric sampling and one charcoal tube sample was collected per chamber per week and analyzed by gas chromatography.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hours/day, 5 days/week
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
2050 mg/m³ (2000 mg/m³)
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
10148 mg/m³ (10000 mg/m³)
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
20324 mg/m³ (20000 mg/m³)
Basis:
analytical conc.
No. of animals per sex per dose:
Group 1 (control): 20 males and 20 females
Group 2 (low): 10 males and 10 females
Group 3 (mid): 10 males and 10 females
Group 4 (high): 20 males and 20 females
Control animals:
yes, sham-exposed

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily for mortality and signs of severe toxic or pharmacologic effects.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed as a group at least once during each exposure. Each animal was removed from its cage and examined twice pretest and once weekly during the study period. Examinations included observations of general condition, skin and fur, eyes, nose, oral cavity, abdomen and external genitalia as well as evaluations of respiration, palpation for tissue masses, circulatory effects, autonomic effects, central nervous system effects, changes in motor activity, and reactivity to handling or sensory stimuli.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were removed from their cages and weighed twice pretest, weekly during treatment and terminally. Terminal, fasted body weights were obtained just prior to necropsy.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

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: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Pretest and at study termination
- Dose groups that were examined: All animals except Genotox and Immunotox animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Interim (~ 4 weeks) and Terminal intervals
- Anaesthetic used for blood collection: Yes - carbon dioxide/oxygen; 60%/40%
- Animals fasted: Yes
- Parameters checked in table were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 4th week interval
- Animals fasted: Yes
- How many animals: All animals
- Parameters checked in table were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Testing was staggered over several sessions and was conducted on non-exposure days or at least 16 hours post-exposure.
- Dose groups that were examined: 10 animals/sex/exposure group
- Battery of functions tested: grip strength / Home cage evaluations/ handling evaluations/ open field evaluations/ reflex assessments/ landing foot splay/ hindlimb extensor strength/ air righting ability/ body weight/ motor activity/ sensory activity

OTHER: Genotoxicity evaluations and Immunotoxicity evaluations
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
The following parameters were analyzed statistically:

mean body weight values and body weight changes (from pretest)
mean feed consumption values (presented as grams of feed/kg of body weight/day)
mean clinical laboratory values
mean organ weights, organ/body weight ratios and organ/brain weight ratios
mean motor activity counts
mean FOB data including forelimb and hindlimb grip strength measurements and mean landing foot splay measurements

Appropriate statistional methods of analysis were conducted.

Results and discussion

Results of examinations

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):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not specified
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY: There was one death and it was considered not treatment-related. The test animals were unremarkable during the exposure periods. A slight increase in red nasal discharge was seen in the 20000 mg/m^3 exposed animals during the 3rd through the 7th weeks of exposures but not during the 4 week recovery period. The effect is considered localized to the site of application of test material.


BODY WEIGHT AND WEIGHT GAIN: There were no toxicologically significant differences in the test substance exposed animals compared to the control animals.


FOOD CONSUMPTION: There were no toxicologically significant differences in the test substance exposed animals compared to the control animals.


HAEMATOLOGY: There were no toxicologically significant differences in the test substance exposed animals compared to the control animals.


CLINICAL CHEMISTRY: There were no toxicologically significant differences in the test substance exposed animals compared to the control animals.


NEUROBEHAVIOUR: The results of the analyses did not indicate a statistically significant exposure-related effect.


ORGAN WEIGHTS: There were no toxicologically significant differences in the test substance exposed animals compared to the control animals.


GROSS PATHOLOGY: No gross abnormalities related to test substance exposure were evident on necropsy examination.


HISTOPATHOLOGY: NON-NEOPLASTIC: Microscopic findings that were considered treatment-related were only found in the nasal turbinates of male and female animals and the kidneys of male animals.

Nasal turbinates: Male and female rats exposed to 20000 mg/m^3 had eosinophilic material within the nasolacrimal duct lumen which correlates with the increase in red nasal discharge noted previously.

Kidneys: Male rats exposed to all three dose levels had eosinophilic hyaline granules within the cytoplasm of renal proximal convoluted tubular epithelial cells. The degree of cytoplasmic granulation varied in an exposure-level dependent manner.

Effect levels

open allclose all
Dose descriptor:
NOAEC
Remarks:
systemic effects
Effect level:
> 20 000 mg/m³ air
Sex:
male/female
Basis for effect level:
other: (exclusive of male hydrocarbon nephropathy)
Dose descriptor:
NOAEC
Remarks:
local effects
Effect level:
10 000 mg/m³ air
Sex:
male/female
Basis for effect level:
other: (red nasal discharge at sign of contact)

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Exposure at all dose levels was associated with hydrocarbon nephropathy in male rats. However, this finding has been generally accepted not to be relevant to human risk assessment. The only other clinically significant finding was slight but reversible increases in red nasal discharge in animals exposed to 20000 mg/m3. If this effect is considered irritation at the site of contact, the systemic NOAEC is to be greater than 20000 mg/m3 and the local NOAEC is 10,000 mg/m3.
Executive summary:

Baseline gasoline vapor condensate was administered by inhalation to Sprague-Dawley rats for 6 hours/day, 5 days/week for 13 weeks at analytical vapor concentrations of 2000, 10000, and 20000 mg/m³ in order to assess subchronic inhalation toxicity. No animals died due to the administration of test material. There were no effects on body weight gain, organ weights, hematology and chemical chemistry analyses, gross pathology or neurobehavior. Microscopic findings that were considered treatment-related were only found in the nasal turbinates of male and female animals and the kidneys of male animals. 13 weeks of exposure of rats to the test substance resulted in slight yet reversible increases in red nasal discharge in animals exposed to 20,000 mg/m³ of vapor. All exposure levels were also associated with hydrocarbon nephropathy in male rats. However, this finding has been generally accepted not to be relevant to human risk assessment. If the red nasal discharge is considered irritation at the site of contact, the systemic NOAEC is determined to be greater than 20,000 mg/m3, and the local NOAEC is 10,000 mg/m3.