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Acute Toxicity: inhalation

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Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: According to or similar to guideline study OECD 403.

Data source

Reference
Reference Type:
publication
Title:
Toxicity of n-C9 to n-C13 alkanes in the rat on short term inhalation
Author:
Nilsen, O.G., Haugen, O.A., Zahlsen, K., Halgunset, J., Helseth, A., Aarset, H., Eide, I.
Year:
1988
Bibliographic source:
Pharmacology and Toxicology 62(5): 259-266

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Deviations:
yes
Remarks:
8 hour exposure
GLP compliance:
not specified
Test type:
acute toxic class method
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
Male Sprague-Dawley rats were obtained from Mollegaard A/S. The animals were acclimatized for 4 to 6 days prior to exposure at a temperature of 22 +/-1 oC, a humidity of 40-70% and a dark-light cycle of 12 hours. The same conditions were used during exposure. Food and water was given ad libitum, except during exposure. At exposure, the weights of animals were within the range of 200g +/- 20%.

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Details on inhalation exposure:
Exposure system. During exposure the animals were kept in conically shaped 0.7 m3 steel chambers with glass front door and walls. Dynamic exposure was performed by passing high oil- and dust- filtered air under pressure through 4 reservoirs of each containing 0.5 1 of the test alkane. At an air flow rate of 30-441 L/min, an outlet concentration equal to 95% of vapour saturation was achieved. The vapour generating system was located in a water bath with a temperature control unit allowing a range of temperature from 0 to 50. Saturation concentrations of alkanes at 2W were achieved by keeping a constant temperature in the bath of 21.6, slightly lower than the temperature in the inhalation chamber. At one occasion (n-nonane, 5280 ppm.) vapour was generated at 22.5°. Air from the vapour generating system was introduced at the top of the exposure chamber and drained from the chamber through a perforated bottom outlet.
Air was withdrawn from the inhalation chambers by a ventilation fan creating a negative pressure of 2-5 mm H20 on the inside of the chamber. During exposure the flow of air through the chambers (exposure and control) was 30-40 L/mm., corresponding approximately to 3 air changes per hour. With a maximum number of 18 animal in each chamber, the volume of animals represented about 0.5% of the total chamber volume. This is a small volume compared to the maximum limit of 5% recommended by WHO. With a volume ratio of 0.5%, 3 air changes per hour was sufficient in order to prevent significant concentrations of ammonia in the inhaled air. The presence of alkane aerosol in the inhalation chamber during exposure was investigated by a Royco mod. 225 particle monitor with an aerosol particle counter sensor mod. For n-tridecane (nC13) the number of aerosol particles in the air with a diameter greater than 0.3 pm was less than 460 per liter. Assuming a mean particle size of 0.7 um, the aerosol n-tridecane concentration in the air would be 1.24 x 10^-7 mg/L. The measured concentration of n-tridecane vapour at saturation was 0.38 mg/L (50 p.p.m.), giving a vapour/aerosol ratio of 3.04 x 10^6.
Analytical verification of test atmosphere concentrations:
yes
Remarks:
Concentration of alkanes in the inhalation chambers was monitored automatically every 15 min by on-line gas chromatography.
Duration of exposure:
8 h
Concentrations:
n-C9 (n-nonane): 5280, 4438, 3560, 2414 ppm
n-C10 (n-decane): 1369 ppm (saturation)
n-C11 (n-undecane): 442 ppm (saturation)
n-C12 (n-dodecane): 142 (saturation)
n-C13 (n-tridecane): 41 ppm (saturation)
No. of animals per sex per dose:
8 animals for each substance at each dose
Control animals:
yes
Details on study design:
The inhalation period was 8 hours at daytime during the light period for all alkanes followed by an observation period of 14 days. For each alkane a high level exposure was performed at its maximum concentration in the air at 20 deg C, i.e. saturated vapour. When this exposure proved lethal, additional exposures were performed in order to determine an exact LC50 value. During inhalation all animals were observed at 15 and 30 mm. intervals. For the first 8 hours after exposure all animals were observed at hourly, then at 2 hours’ intervals during daytime for 14 consecutive days. Groups of 8 animals were exposed for determination of alkane concentrations in blood and brain. Control groups of 4 animals were exposed simultaneously but only to air under otherwise identical conditions. Measurements were performed in 5 surviving animals from the exposed group and in 4 control animals. Groups of 10 animals were included in the pathological study. All were subjected to a complete necropsy which included examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents according to the OECD procedures (OECD 1987). Animals dying during exposure to the saturated air concentration of alkanes at 20 deg C were immediately examined by autopsy, while animals surviving this concentration were killed and examined after the observation period of 14 days. A maximum of 5 exposed animals were investigated for each alkane except for the n-nonane exposure where all 10 animals were examined. The control group for each alkane exposure consisted of 3 rats, giving a total number of 15 control animals.

Determination of alkane concentration in biological material. Concentrations of alkanes were determined in blood and brain via head space gas chromatography immediately after the end of the 8 hour exposure period. Animals were removed from the chamber one by one for immediate decapitation and sample preparation. The preparation of samples was performed after a standardized time schedule with less than two mi between decapitation and isolation of blood and the brain samples from each animal. Immediately after decapitation 2 ml of blood was collected from each animal in heparinized (1,000 IU) glass tubes and rapidly transferred to 15 mln head space vials which were closed by a screw cap with a teflon-faced neoprene septum. After equilibrating the control, “exposed” and standard (0.5—1000 mg/L) samples at 60 for 1 hour during continuous agitation, a 0.1 ml head space sample was taken with a pre-warmed gas-tight syringe and injected onto the gas chromatograph. The gas chromatographic conditions were identical to the conditions described previously for the measurement of alkane concentrations in the inhaled air. After decapitation and blood collection the brain was excised, weighed, and homogenized in 4 volumes of 0.25 M sucrose at 4 in a Potter-Elvehjem homogenizer. Two ml of homogenate was transferred to head space vials and further treated as described above, loss of alkanes during the sampling procedures represented no significant analytical source of error as demonstrated in recovery studies with n-nonane, the most volatile of the test substances.

Determination of LC50. The LC50 value was determined by 8 hour exposure and an observation period of 14 days. Groups of 10 animals were exposed to 4 different concentrations of alkanes, the highest concentration produced close to a 100% mortality. The lowest concentration and two intermediate concentrations reported no cases of death.

Behavioural effects. In addition to general activity, acute effects as coordination difficulties, tremor, spasms and lethality were monitored.

Fixation procedures. For histological examinations the entire brain, both lungs, the liver, the heart and the kidneys were initially fixed for 12 hours in 10% formol ethanol containing 5% glacial acetic acid. Pieces of each organ were then transferred to 70% ethanol and processed for paraffin embedding. Sections were cut with the microtome knife. Sections from each organ were stained in batches with haematoxylin-eosin-saffron.

Light microscopic evaluation. After staining, the slides were given random numbers. Each slide was studied by a group of 4 pathologists working in pairs. One transverse section of the cerebrum, one sagittal section of the cerebellar vermis, one section of each lung and one section of the heart, the liver and the right kidney were evaluated for each animal.

Morphometry. Morphometry was carried out in order to quantify the loss of cerebellar Purkinje cells in the animals exposed to nonane.
Statistics:
For a detailed description of the morphometrical procedure, see Weibel (1979).
Weibel, E. K.: Stereological methods. Vol. I. Academic Press. London 1979.

Results and discussion

Effect levelsopen allclose all
Sex:
male
Dose descriptor:
other: NOAEC
Effect level:
2 414 ppm
Exp. duration:
8 h
Remarks on result:
other: for all materials tested (n-C9 to n-C13 alkanes)
Sex:
male
Dose descriptor:
LC50
Effect level:
4 467 ppm
Exp. duration:
8 h
Remarks on result:
other: n-nonane
Sex:
male
Dose descriptor:
LC50
Effect level:
> 1 369 ppm
Exp. duration:
8 h
Remarks on result:
other: n-decane; maximum attainable concnetration
Sex:
male
Dose descriptor:
LC50
Effect level:
> 442 ppm
Exp. duration:
8 h
Remarks on result:
other: n-undecane; maximum attainable concnetration
Sex:
male
Dose descriptor:
LC50
Effect level:
> 142 ppm
Exp. duration:
8 h
Remarks on result:
other: n-dodecane; maximum attainable concnetration
Sex:
male
Dose descriptor:
LC50
Effect level:
> 41 ppm
Exp. duration:
8 h
Remarks on result:
other: n-tridecane; maximum attainable concnetration
Mortality:
No deaths were noted for the maximum vapor concentration for the n-C10 to n-C13 alkanes tested. For n-nonane (n-C9) the following deaths were observed: 5280 ppm - 9/10; 4438 ppm - 4/10; 3560 ppm - 1/10; 2414 ppm - 0/10.
Clinical signs:
A sedative effect or narcosis was not observed. Neither deaths nor adverse behavioral effects were observed during 8 hours exposure to n-decane n-undecane, n-dodecane, n-tridecane, and n-nonane (2414 ppm). Symptoms such as tremor, spasms and limb paralysis were observed in higher concentrations of n-nonane.
Body weight:
There was a significant body weight increase in the 4438 ppm exposed animals. No other body weight changes were noted.
Gross pathology:
No significant differences in the weight of the heart, kidneys, liver, or brain were demonstrated between the groups. No morphological alterations were observed in heart or kidneys.
Other findings:
Microscopically, dilatation of the sinusoids was found in all four animals dying during exposure to n-nonane and three of these animals also showed definite though slight fatty changes of the liver cells. In animals surviving exposure to n-nonane and in animals exposed to other alkanes, and among the controls, no such changes were observed.

Lungs. The total weight of the lungs in two of the animals dying during exposure to n nonane was approximately twice the weight of the controls 2.86 g and 2.88 g, respectively. Among the other animals exposed to various other alkanes, no weight alterations were observed. Microscopically, three animals showed marked pulmonary edema. These animals died during exposure to n-nonane and in two of them the pulmonary weight was also increased. All animals exposed to 4438 ppm n-nonane showed a blue discoloration of the skin during exposure, giving the impression of peripheral cyanosis, and thus cardiopulmonary insufficiency.

Brain. No macroscopic abnormalities were recorded in any of the animals. No pathological changes were found in the large brain in any of the animals exposed to the series n-C10 to n-C13 or of their respective controls. In animals exposed to n-nonane (4438 ppm) and which died during exposure, pathological changes were absent. Among the six animals surviving for 14 days after exposure, one animal showed a few severely damaged neurons of the hippocampus. In the cerebellar cortex no alteration could be demonstrated among animals exposed to n-C10 to n-C13 or their controls or in animals dying acutely during exposure to n-nonane or their controls. However, in n-nonane (4438 ppm treated group) exposed animals surviving for 14 days after exposure, extensive changes were observed including rarification of Purkinje cells and in some instances also a high number of severely damaged neurons were observed. These changes apparently were not entirely random but seemed to some extent to be segmental. In areas showing massive loss of Purkinje cells there was no obvious glial reaction.

Morphometry of the cerebellum. The results demonstrate clearly a loss of Purkinje cells in the animals which survived exposure to 4438 ppm n-nonane. This is reflected by a reduced density of Purkinje cell profiles along the line which defines the Purkinje cell layer (N0). The reduced density of the visible transections is only in part explained by a reduction of the cell size, as there are also a significantly reduced number of Purkinje cells per unit area of Purkinje cell layer (Ns). The results also indicate a reduction of tissue volume in the other layers of the cerebellum as there was a decrease in cerebellar volume relative to the extent of the Purkinje cell layer. There was no clear-cut change of the volume distribution between the different layers. There was no detectable difference between the animals which died during exposure and the controls.

Any other information on results incl. tables

Table 1: Concentration of alkanes in rat blood and brain at the end of an 8 hour exposure period


  Exposure Conc. (ppm) Blood concentration (mg/L) Brain concentration (mg/kg) Blood/air ratio Blood/air ratio

n-C9 (n-nonane)

5280 238* 1136* 8.6 41.3
  4438 109 919 4.7 39.5
  3560 135 590 7.2 31.7
  2414 57 314 4.5 24.3
n-C10 (n-decane) 1369 (saturation) 542 239.7 6.8 29.9
n-C11 (n-undecane) 442 (saturation) 11.7 45.4 4.1 16
n-C12 (n-dodecane) 142 (saturation) 3.3 4.2 3.3 4.3
n-C13 (n-tridecane) 41 (saturation) 0.84 < 0.5 1.6 <1.6

 

*Concentration measured in one animal that died 0.5 hour before the end of the exposure period.

Applicant's summary and conclusion

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The LC50 after an 8 hour exposure for acute inhalation exposure to n-C10 to n-C13 alkane vapors is greater than the highest obtainable vapor concentration. The LC50 after an 8 hour exposure for n-C9 alkane vapors was determined to be 4467ppm. For n-C9 to n-C13 alkanes, classification as an acute inhalation toxicant is not warranted under the new Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP) or under Directive 67/548/EEC for dangerous substances and Directive 1999/45/EC for preparations.
Executive summary:

Individual alkanes (n-C9 to n-C13) were administered via individual inhalation chambers to eight Sprague-Dawley rats at their respective maximum attainable vapor concentration (n-C9: 5280, 4438, 3560, 2414 ppm; n-C10: 1369 ppm; n-C11: 442 ppm, n-C12; 142 ppm; n-C13: 41 ppm) for eight hours to assess acute inhalation toxicity. There was no mortality and no gross pathological alterations noted in any of the animals treated with n-C10 to n-C13.  The LC50 after an 8 hour exposure for n-C9 alkane vapors was determined to be 4467ppm.  The LC50 after an 8 hour exposure for acute inhalation exposure to n-C10 to n-C13 alkane vapors is greater than the highest obtainable vapor concentration. The LC50 after an 8 hour exposure for n-C9 alkane vapors was determined to be 4467ppm. For n-C9 to n-C13 alkanes, classification as an acute inhalation toxicant is not warranted under the new Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP) or under Directive 67/548/EEC for dangerous substances and Directive 1999/45/EC for preparations.