Registration Dossier

Toxicological information

Repeated dose toxicity: inhalation

Currently viewing:

Administrative data

chronic toxicity: inhalation
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented publication similar or equivalent to OECD 453.

Data source

Reference Type:
A Chronic Inhalation Study with Unleaded Gasoline Vapor
MacFarland et al.
Bibliographic source:
Journal of the American College of Toxicology. Vol.3, No. 4

Materials and methods

Test guideline
equivalent or similar to guideline
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
GLP compliance:
not specified
Limit test:

Test material

Constituent 1
Reference substance name:
Cas Number:
Constituent 2
Reference substance name:
Unleaded motor gasoline
Unleaded motor gasoline
Test material form:
other: low viscosity liquid hydrocarbon
Details on test material:
The unleaded automotive motor fuel (gasoline) used in the study was prepared to conform with the specifications of unleaded gasoline in use in the US in 1976, as determined by a road octane survey (DuPont Road Octane Survey, summer 1976; At the Lime the gasoline was blended for the study, benzene concentrations in US gasolines averaged about 1%, with a maximum approaching 2%; therefore, benzene content of the gasoline was adjusted to the upper limit of US gasolines. The specifications are shown in Table I, but more detailed information on chemical composition is provided in the Appendix.

The following details are found in the attachment:
Reid vapour pressure
distillation range
research octane number
motor octane number

Test animals

other: rat and mouse
other: Fischer 344 and B6C3F
Details on test animals or test system and environmental conditions:
TEST ANIMALS: Rats STRAIN: Fischer 344 SEX: male/female
- Age at study initiation: 6 weeks
- Weight at study initiation: M: 95-129 g; F: 79-105 g
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 weeks

TEST ANIMALS: Mice STRAIN: B6C3F SEX: male/female
- Age at study initiation: 6 weeks
- Weight at study initiation: M: 14-26 g; F: 12-20 g
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 weeks

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
other: unchanged (no vehicle)
Details on inhalation exposure:
- Exposure apparatus: 16 m^3 stainless steel and glass exposure chambers
- System of generating particulates/aerosols: Gasoline was delivered from a liquid metering pump to a heated countercurrent vaporization column and completely volatilized. Dry nitrogen at 5-6 L/min was used to carry the vapor into the main inlet pipe of the chamber.
- Temperature, humidity in air chamber: Measured each day at the start of exposure and at 1, 3, and 5 hours
- Air flow rate: Between 900 and 1900 L/min
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Nominal concentrations were determined daily, and calculations of concentration in ppm were made by using weight loss data and assuming an average molecular weight of 108 g/mol for the gasoline.

Analytical concentrations were determined by drawing samples from the chambers into a gas chromatograph equipped with a flame ionization detector.
Duration of treatment / exposure:
Male rats: 107 weeks
Female rats: 109 weeks
Male mice: 107 weeks
Female mice: 113 weeks
Frequency of treatment:
6 hours per day, five days per week
Doses / concentrationsopen allclose all
Doses / Concentrations:
322 mg/m3
analytical conc.
Doses / Concentrations:
1402 mg/m3
analytical conc.
Doses / Concentrations:
9869 mg/m3
analytical conc.
No. of animals per sex per dose:
100 animals per sex per dose
Control animals:
yes, sham-exposed


Observations and examinations performed and frequency:
- Time schedule: Twice daily

- Time schedule: once a month

- Time schedule for examinations: Body weights were recorded monthly for the first 17 months and biweekly thereafter.

- Time schedule for collection of blood: 18-month interim and terminal sacrifice
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 7 male and 7 female rats per group
- Parameters checked: hemoglobin, hematocrit, erythrocyte count, total and differential leukocyte count, platelet count, reticulocyte count, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration.

- Time schedule for collection of blood: At the interim sacrifices: 3, 6, 12, and 18 months
- Animals fasted: Yes
- How many animals: 7 male and 7 female rats per group
- Parameters checked: alkaline phosphatase, glutamic oxalacetic transaminase, glutamic pyruvic transaminase, ornithine carbamyl transferase, and isocitrate dehydrogenase


Sacrifice and pathology:
Body weight, hematologic, and serum biochemical data were tested for homogeneity of variance, followed by a parametric analysis of variance. When a significant F-ratio was obtained, individual group comparisons were performed, utilizing Student's T-test when variances were heterogeneous and Dunnett's test when homogeneous. In some cases where the number of animals was small and the variance heterogeneous, the non-parametric multiple-group test of Kruskal-Wallis was applied, and where appropriate, individual group comparisons were made with the Mann-Whitney U test.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
effects observed, treatment-related
Details on results:
CLINICAL SIGNS AND MORTALITY: No dose-related differences were noted in spontaneous death rate in either species.

BODY WEIGHT AND WEIGHT GAIN: Male rats in the high dose group had significantly lower body weights than controls from week 13 to termination. Female rats in the high dose group showed a similar depression, which was significant from week 26 to the end of the study. Male mice receiving the high dose exhibited a lower body weight than controls, which was significant from week 66 to termination.

HAEMATOLOGY: No treatment-related effects in either species.

CLINICAL CHEMISTRY: No treatment-related effects in either species.

ORGAN WEIGHTS: The kidney weights of male rats in the high dose group were elevated from the 3 month interim sacrifice through termination. At termination, the relative kidney weights of male rats dosed with 1402 mg/m3 and female rats dosed with 9869 mg/m3 were also elevated. There was a dose-related relative increase in the testes and ovaries of the rats receiving mid- and high-doses, and slight depression in absolute heart weights was noted in the hige dose groups (male and female rats). In mice, there were no treatment-related effects.

GROSS PATHOLOGY: A compound-related increase in liver nodules and masses was seen in female mice exposed to the high level.

HISTOPATHOLOGY: NON-NEOPLASTIC: Both sexes of rats exhibited a mild, multifocal, pulmonary inflammatory response characterized by an accumulation of alveolar macrophages in the alveolar speces of the lungs. The incidence of these aggregates of macrophages was similar in all treated group s and in control animals and were not considered to be treatment-related.

MICE: Female mice treated with 9869 mg/m3 gasoline vapor exhibited an increased incidence of hepatocellular carcinoma, considered to be treatment-related. The tumors were of 2 types: hepatocellular adenomas and hepatocellular carcinomas. Two female mice in the high dose group developed renal tumors.
RATS: Male rats exhibited primary renal neoplasms (1, 6, and 7 in low, mid and high dose groups, respectively) after 18 month exposure. Reexamination of the kidney sections some years after the initial reading revealed an increase in the incidence and severity of regenerative epithelial changes, and dilated tubules containing proteinaceous material. This findings are consistent with alpha 2 microglobulin-induced nephropathy.

Effect levels

Dose descriptor:
Effect level:
1 402 mg/m³ air (analytical)
Basis for effect level:
other: Based on decreased body weight gain in mice and rats at 9869 mg/m3.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

The NOAEC for unleaded gasoline vapor is 1402 mg/m3 under the test conditions of this study, based on decreased body weight gain in mice and rats at 9869 mg/m3.
Executive summary:

Unleaded gasoline vapor was administered by inhalation to Fischer 344 rats and B6C3F mice for 6 hours/day, 5 days/week for up to 113 weeks at analytical vapor concentrations of 322, 1402, and 9869 mg/m3to assess inhalation toxicity. No consistent, compound-related changes were seen in mortality, hematology or clinical chemistry parameters in either species. Significant depression of body weight gain was seen in both sexes of rats and male mice exposed to the highest level of gasoline vapor. On gross necropsy, a compound-related increase in liver nodules and masses was seen in female mice exposed to the highest dose level. In addition, male rats exposed to gasoline vapor at all concentrations exhibited primary kidney neoplasms, correlated histopathologically with an increase in the incidence and severity of regenerative epithelial changes and dilated tubules containing proteinaceous material. The kidney effects observed in male rats are indicative of alpha-2u-globulin nephropathy. Alpha-2u-globulin nephropathy, also known as hyaline droplet nephropathy, results from the formuation of complexes with a naturally occurring protein (alpha-2u-globulin) in the kidneys of male rats. These complexes can accumulate in the proximal renal tubule and may produce species-specific histopathological changes. Based on the decreased body weight gain at the highest dose, the no observed adverse effect concentration (NOAEC) was 1402 mg/m3.