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Toxicological information

Neurotoxicity

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Administrative data

Endpoint:
neurotoxicity: sub-chronic inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable well-documented publication which meets basic scientific principles.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1993

Materials and methods

Principles of method if other than guideline:
Groups of 20 male rats were exposed via inhalation to 100, 500, or 1500 ppm high flash aromatic naphtha for 6 hrs per day, 5 days per week, for 13 weeks. Neurotoxicity testing was performed at 5, 9, and 13 weeks of exposure. Animals were evaluated for motor activity, fore and hind limb grip strength, audio startle response, thermal response, and hind limb foot splay. Animals were also observed weekly for clinical and behavioural signs, and pharmacotoxic signs.
GLP compliance:
yes
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Portage, MI
- Age at study initiation:52 days
- Weight at study initiation: 300 g
- Housing: Inidividually housed in wire mesh cages
- Diet (e.g. ad libitum): Purina Certified Rodent Chow No. 5002 ad libitum except during exposure
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2-3 weeks


ENVIRONMENTAL CONDITIONS
Animal husbandry followed standards by the US Department of Health, Education, and Welfare (1985)

Administration / exposure

Route of administration:
inhalation: vapour
Details on exposure:
- System of generating particulates/aerosols: Test atmosphere was generated by heating nitrogen to 200°C by passing it through a 1 l stainless steel cylinder with a 1500 W band heater. The nitrogen then passed through a glass column 7.6 cm diameter and 30 cm long packed with glass beads. Test material was delivered by a metering pump into Teflon tubing, to the bottom of the column. The liquid test substance vaporized as it went up the column with the nitrogen. The vapor then went into the test chambers where dilution with the chamber ventilation air produced the desired concentrations.
TEST ATMOSPHERE
- Brief description of analytical method used: Measurements made hourly using gas-phase IR.
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration of the test material in the test chambers was determined by GC analysis.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hours/day 5 days/week
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
101 (2.5) ppm
Basis:
analytical conc.
target conc.: 100 ppm
Remarks:
Doses / Concentrations:
432 (2.8) ppm
Basis:
analytical conc.
target conc.: 500 ppm
Remarks:
Doses / Concentrations:
1320 (13) ppm
Basis:
analytical conc.
target conc.: 1500 ppm
No. of animals per sex per dose:
20 male animals per group
Control animals:
yes, concurrent no treatment

Examinations

Observations and clinical examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: pretest, monthly intervals
- Cage side observations checked: tremors, convulsions, lacrimation, respiration, urination, defecation, vocalization, piloerection, pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: weekly


Neurobehavioural examinations performed and frequency:
FUNCTIONAL OBSERVATIONAL BATTERY: Yes
- Parameters examined: Fore and hind leg grip strength, thermal response, hind limb foot splay
- Description of procedures: Fore and hind leg grip strength: Force transducers (Grass Instrument Co. Model FT-10) were connected to a chart recorder and mounted on a plexiglass platform. Animals were tested three times and the average used.
Thermal response: An Omnitech, Inc. analgesiometer was used for the hot plate stimulus test. Hind limb foot splay: Diluted tatoo ink was placed on the fifth toe of each hind foot, and the animals dropped from a standard height.
- Time schedule for examinations: 5, 9, 13 weeks after exposure.

LOCOMOTOR ACTIVITY: Yes
- Replicates used: 18-20 animals
- Type of equipment used: Digiscan Activity Meter
- Length of session, number and length of subsessions: Total session time was 0-30 minutes, 0-2 minutes acclimation, then 2-5, and 5-minute intervals
- Total activity: Activity was measured using vertical and horizontal infrared beams. Vertical and horizontal activity was recorded seperately each time an animal broke a beam.

AUDITORY STARTLE REFLEX HABITUATION: Yes
- Number of animals: 20 per dose
- Days of testing: 5, 9 and 13 weeks after exposure initiation
- Type of equipment used: Black acrylic startle box fabricated in the laboratory. The floor of the box was supported by a load cell. Rats were placed in the box, and after five minutes, were stimulated using white noise.
- Number of trials performed: 10
- Length (msec) and intensity (dB) of sound: 110 dBA, 50 msec
- Length of interval between trials: 15 seconds
- Other procedural details: The 2nd through 11th stimuli were measured for amplitude (kg) and latency (ms).
Sacrifice and (histo)pathology:
- Time point of sacrifice: 13 weeks after start of exposure
- Number of animals sacrificed: 10 per group
- Procedures for perfusion: After intraperitoneal anesthesia with barbiturates, heart was perfused with heparizined saline followed by glutaraldehyde, and paraformaldehyde
- Tissues evaluated: proximal sciatic nerves and extensions, tibial, peroneal, and sural nerves, L3 and L4 dorsal root ganglia, dorsal and ventral root fibers, spinal cord, forebrain, cerebrum, midbrain, cerebellum, pons, medulla
- Type of staining: luxol fast blue, hematoxylin, eosin
- Embedding media: osmium tetraoxide
- Number of sections: two
- Number of animals evaluated from each sex and treatment group: 10 animals per group
Statistics:
Kruskal-Wallis one-way analysis of variance, followed if needed by the Mann-Whitney U using a Bonferroni correction were used for analysing body weight, motor activity, and the functional observational battery. Parametric statistics, analysis of variance on the square root activity.

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS AND MORTALITY
No animals died during the study. No clinical signs were considered exposure related.

BODY WEIGHT AND WEIGHT GAIN
The 1500 ppm had depressed weight gain throughout the study. The 500 ppm group also had depressed weight gain, but the difference was only significant at 4 weeks.


NEUROBEHAVIOUR
No signicant differences in motor activity were found in any group. No differences in hind foot splay and grip strength were noted in any group at any time. The only significant difference in auditory response time was in the 500 ppm group at week 9. Since this finding was not dose-related, it was considered to not be biologically important. A significant difference in the thermal response in the mid-dose groups, 100 and 500 ppm, was seen in the pre-test. However, this was most likely due to an unusually low response time for the control group at this time-point.

NEUROPATHOLOGY
No exposure related neuropathological lesions, or degenerative changes were seen.

Effect levels

open allclose all
Dose descriptor:
NOAEC
Effect level:
1 500 ppm
Sex:
male
Basis for effect level:
other: The test substance had no neurotoxic effects at any of the test concentrations.
Remarks on result:
other:
Dose descriptor:
LOAEC
Effect level:
1 500 ppm
Sex:
male
Basis for effect level:
other: The test substance was slightly toxic at 1500 ppm as evidenced by depressed weight gain.
Remarks on result:
other:

Applicant's summary and conclusion

Conclusions:
The test substance was not neurotoxic at any concentration in the test, therefore the NOAEC = 1500 ppm for neurotoxicity. There was a slight depression in weight gain at 1500 ppm, therefore the LOAEC = 1500 ppm for sub-chronic inhalation toxicity.
Executive summary:

This study was conducted to determine the neurotoxicity of high flash aromatic naphtha. Groups of 20 male rats were exposed via inhalation to 100, 500, or 1500 ppm high flash aromatic naphtha for 6 hrs per day, 5 days per week, for 13 weeks. Neurotoxicity testing was performed at 5, 9, and 13 weeks of exposure. Animals were evaluated for motor activity, fore and hind limb grip strength, audio startle response, thermal response, and hind limb foot splay. Animals were also observed weekly for clinical and behavioural signs, and pharmacotoxic signs. Results of the neurotoxicity testing showed no neurotoxic effects giving an NOAEC of 1500 ppm for neurotoxicity. The 1500 ppm group showed a 12% reduction in body weight gain. Based on the reduction in weight gain, the LOAEC for the test substance is 1500 ppm for sub-chronic inhalation toxicity.