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Neurotoxicity

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Description of key information

In a neurotoxicity study, 32 male Sprague-Dawley rats were exposed to room air, 1000 mg/m3(1 mg/L) of JP-8 jet fuel or 1200 mg/m3(1.2 mg/L) of JP-5 jet fuel 6 hours a day, 5 days a week, for 6 weeks (30 exposures). After 65 days of recovery, rats were subjected to different neurobehavioral test to evaluate changes in performance capacity.

Results indicate that exposure to JP-5 or JP-8 vapour does not affect basic sensory, motor, or inhibitory functions. Nor does exposure affect the capacity of rats to learn and recall tasks of minimum complexity. The NOAEC is 1000 mg/m3for JP-8 and 1200 mg/m3for JP-5.

Key value for chemical safety assessment

Effect on neurotoxicity: via oral route

Endpoint conclusion
Endpoint conclusion:
no study available

Effect on neurotoxicity: via inhalation route

Link to relevant study records
Reference
Endpoint:
neurotoxicity: short-term inhalation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This study is classified as reliable with restrictions because it is an acceptable and well-documented study report.
Justification for type of information:
Read across justification included in Section 13
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
no guideline followed
Principles of method if other than guideline:
Male rats were exposed to a single concentration of JP-5 or JP-8 for 6 hours a day, 5 days a week, for 6 weeks (30 days of exposure). Animals were then left untreated for 65 days. At the end of the 65 days, the rats were evaluated for 10 different neurobehavioral tests over the next 21 days. Rats were sacrificed with 28 hours of completing the tests and necropsied.
GLP compliance:
not specified
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Raleigh, North Carolina
- Age at study initiation: 64 days old
- Weight at study initiation: Not reported
- Housing: Individually in hanging polycarbonate shoebox cages
- Diet (e.g. ad libitum): ad libitum except during exposure
- Water (e.g. ad libitum): ad libitum except during exposure
- Acclimation period: 14 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 to 25
- Humidity (%): 40 to 70%
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light


Route of administration:
inhalation: vapour
Vehicle:
air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 270-L Hinners-type whole-body inhalation chambers
- Method of holding animals in test chamber: Individual housing spaces (17 x 12 x 17 centimetres)
- Source and rate of air: Counter current room air source at a rate of 70 +/- 3 L/min
- Temperature, humidity, pressure in air chamber: Although these parameters were specified as being measured in the exposure chamber, details were not provided in the report.
- Air flow rate: 70 +/- 3 L/min
- Air change rate: Not reported


TEST ATMOSPHERE
- Brief description of analytical method used: Concentrations were quantified at 20-minute intervals using an infrared absorbance spectrophotometer (absorbance band between 3.4 and 3.5 micrometres calibrated to the known mass concentration of hexane). Gas chromatographic analysis of the fuel, fuel vapour, and spent fuel were also performed.
- Samples taken from breathing zone: No


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
There was no quantifiable aerosols detected during the daily sampling. With the exception of a couple of days the concentration of JP-8 vapour was 1000 mg/m3 +/- 10% and the concentration of JP-5 vapour was 1200 mg/m3 +/- 10%. For JP-8, day 1 had a mean exposure concentration of 1650 mg/m3. For JP-5, day 14 and 15 had mean concentrations of 876 and 995 mg/m3, respectively.
Duration of treatment / exposure:
6 weeks
Frequency of treatment:
6 hours a day, 5 days a week
Remarks:
Doses / Concentrations:
JP-5: 1200 mg/m3; JP-8: 1000 mg/m3
Basis:
nominal conc.
No. of animals per sex per dose:
32 males per treatment group
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: Not reported
- Rationale for animal assignment (if not random): Although animals were randomly assigned to groups, but were semi-randomized to daily placement in the exposure chamber.
Observations and clinical examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No data


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Not reported


BODY WEIGHT: Yes
- Time schedule for examinations: Not reported


FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data



WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No


OPHTHALMOSCOPIC EXAMINATION: No


Specific biochemical examinations:
NEUROPATHY TARGET ESTERASE (NTE) ACTIVITY: No


CHOLINESTERASE ACTIVITY: No


OTHER: The concentration of norepinephrine, dopamine, 3,4-dihydroxyphenylacetatic acid, homovanillic acid, serotonin, and 5-hydroxyindoleacetic acid were measured in the supernatants of brain tissue and blood.
Neurobehavioural examinations performed and frequency:
FUNCTIONAL OBSERVATIONAL BATTERY: Yes
- Parameters measured included forelimb grip strength, tail flick response,
- Description of procedures: Forelimb grip strength: Hand-held rats were allowed to grasp a mesh pull screen of a Standard Grip Strength Meter for Rats, then were slowly pulled, three trials were conducted; tail flick response: rats were placed in a restrainer and tails were exposed to heat from a bulb located under the apparatus, the timer was stopped when the tail flicked, three trials were conducted
- Minimization of bias:
- Same technicians used throughout testing: No data
- Technicians were blind to treatment status of animals: No data

LOCOMOTOR ACTIVITY: Yes
- Type of equipment used: Animal Activity Meters
- Length of session, number and length of subsessions: 30 minutes
- Parameters measured: Total horizontal beam interruptions, total vertical beam interruptions, total time ambulatory, total time resting, total time in stereotypic activity, total time circling, and total time in forward locomotion


AUDITORY STARTLE REFLEX HABITUATION: Yes
- Number of animals: All animals
- Days of testing: Over a 21 day testing period beginning 65 days after exposure
- Type of equipment used: Acoustic Startle Response System (model SR-Lab) restraint tubes
- Environmental conditions: Chambers were illuminated with 40-W standard light bulbs. Rats were placed within the restraint tubes so that their heads were oriented in the direction of an audio speaker located approximately 3 centimetres from the restraint tube door.
- Number of trials performed: A single session consisted of 8 presentations
- Length (msec) and intensity (dB) of sound: 100 msec; 74 dB as baseline control and 105 dB for startle pulse
- Length of interval between trials: 10 seconds


LEARNING AND MEMORY TESTING: Yes, appetitive reinforcer approach sensitization
(1) Overall testing design
- Number of animals: All animals
- Days of testing: 3 days
- Evaluation of both short and long term recall: no
(2) Equipment used
- Type of equipment: Plexiglas rectangular chamber with floor covered in black contact paper; in each corner was a fluorescent light fixture grating (a triangular-shaped stimulus display area), semicircles were etched around the stimulus area to define the rat approach to stimulus
- Environmental conditions:
(3) Testing and training procedures
- Number of trials per day: One
- Number of days of testing: Three
- Stimulus parameters: Raw hamburger
- Learning criteria: After 2 days of conditioning with raw hamburger, rats were placed in the centre of the chamber facing either the wall with the two baited stimuli or the nonbaited wall, observers measured the number of seconds that rats spent with two paws on or across the semicircle lines and the number of seconds spent in the stimulus approach zones near the two nonbaited stimulus display areas.


LEARNING AND MEMORY TESTING: Yes, passive avoidance
(1) Overall testing design
- Number of animals: All animals
- Days of testing: Two
- Evaluation of both short and long term recall: no
(2) Equipment used
- Type of equipment (including manufacturer, if available): Two-Chamber Shuttle Box (Reflex-16 Shuttle Box, Columbus Instruments)
(3) Testing and training procedures
- Number of trials per day: One
- Number of days of testing: Two
- Changes across days: No
- Inter-trial intervals: 24 hours
- Use of cut-off times or error correction procedures: 600 seconds
- Learning criteria: Animals were shocked upon entering the nonilluminated chamber. Amount of time to enter nonilluminated chamber was measured on the both days.

LEARNING AND MEMORY TESTING: Yes, porsolt forced swim test
(1) Overall testing design
- Number of animals: All animals
- Days of testing: 2 days
- Evaluation of both short and long term recall: no
(2) Equipment used
- Type of equipment (including manufacturer, if available): Plexiglas cylinders filled with temperature-controlled water
- Environmental conditions: Water was maintained at 25 to 27 degrees Celsius and the water was deep enough that the rats could not touch the bottom without being submerged
(3) Testing and training procedures
- Number of trials per day: One
- Number of days of testing: Two
- Changes across days: No
- Inter-trial intervals: 24 hours
- Use of cut-off times or error correction procedures: 15 minutes or 5 seconds of submersion
- Parameters measured included: time to initial immobility response (10 consecutive seconds of immobility), time swimming actively, time immobile, time spent diving, and number of body revolutions

LEARNING AND MEMORY TESTING: Yes, Morris Water Maze
(1) Overall testing design
- Number of animals: All animals
- Days of testing: Two
- Evaluation of both short and long term recall: no
(2) Equipment used
- Type of equipment (including manufacturer, if available): Morris Water Maze
- Environmental conditions: Cylindrical metal tank filled with water maintained at 25 to 27 degrees Celsius
(3) Testing and training procedures
- Number of trials per day: One
- Number of days of testing: Two
- Changes across days: No
- Inter-trial intervals: 24 hours
- Use of cut-off times or error correction procedures: Submersion for more than 5 seconds
- Learning criteria: Rats were placed as far from the escape platform as possible and the time to locate the platform, distance travelled, time in apparatus quadrant containing platform, time "wall" swimming, time circling, and time immobile were measured on each day and compared.

Sacrifice and (histo)pathology:
- Time point of sacrifice: 86 days after exposure ended
- Number of animals sacrificed: All animals
- Parameters measured:
- Brain weight: No
- Length and width of brain: No
- Other: Neurotransmitter and metabolite concentrations
- Procedures for perfusion: None
- Tissues evaluated: Tissues were not histologically evaluated.
Other examinations:
A conspecific approach test was also conducted where individually housed rats were allowed to interact with other rats. Rats were placed in 2 adjacent clear plastic shoebox cages with ports cut into each end of the cages for 10 minutes. Social ports were placed within 2 centimetres of each other and nonsocial ports were placed 78 centimetres from each other. The frequency of nose-pokes in each port were measured.
Statistics:
Student's t-test or one-way analysis of variance with post hoc Fischer's least significant difference. For neurotoxicity tests that had scores limited by a maximum or "cap" score or data that was not normally distributed, a Mann-Whitney U-test was performed.
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):
not specified
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Clinical biochemistry findings:
effects observed, treatment-related
Behaviour (functional findings):
effects observed, treatment-related
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY: There were no treatment-related effects on clinical signs or mortality.


BODY WEIGHT AND WEIGHT GAIN: There were no treatment-related effects on body weight.


BIOCHEMISTRY: Serum levels of 5-hydroxyindoleacetic acid were significantly increased in JP-5 treated rats (approximately 206% of control), but were significantly decreased in JP-8 treated rats (approximately 38% of control). No other changes were noted in blood biochemistry. There was a significant increase in dopamine levels in the hippocampus (146% of control) and dihydroxyphenylacetic acid (a dopamine metabolite) in the cerebral cortex (146% of control) of JP-5 treated rats compared to the controls. There was a significant decrease in 5-homovanillic acid (a serotonin metabolite) in the hippocampus (66% of control) of JP-5 treated rats compared to the controls. JP-8 exposed rats had significantly decreased levels of dihydroxyphenylacetic acid in both the cerebellum (64% of control) and brainstem (82% of control) regions.


NEUROBEHAVIOUR: There were no treatment-related effects on acoustic startle response, prepulse inhibition of acoustic startle response, total locomotor activity, tail flick response, nonspecific approach, porsolt forced swim test, passive avoidance, or Morris Water Maze. In the appetitive stimulus approach sensitization test, there was an increase in the seconds to approach the novel appetitive stimulus in both JP-5 (75 seconds compared to 40 seconds in controls) and JP-8 (110 seconds compared to 55 seconds n controls) treated rats; however, only JP-8 treatment resulted in a significant increase. There was no difference in the amount of time spent in the unbaited stimulus areas or in total line-crossing measures. JP-5 treated rats had a significant increase in forelimb grip strength compared to the controls for all three successive trials (approximately 122% to 155% of control).



Basis for effect level:
other: see 'Remark'
Remarks on result:
not measured/tested
Remarks:
Effect level not specified
Conclusions:
There were some changes in neurotransmitter or metabolite concentrations in the serum and brain in the treated rats, but results were not consistent between the two types of fuel. The results indicate that exposure to JP-5 or JP-8 vapour does not affect basic sensory, motor, or inhibitory functions. Nor does exposure affect the capacity of rats to learn and recall tasks of minimum complexity.
Executive summary:

In this neurotoxicity study, 32 male Sprague-Dawley rats were exposed to room air, 1000 mg/m3 (1 mg/L) of JP-8 jet fuel or 1200 mg/m3 (1.2 mg/L) of JP-5 jet fuel 6 hours a day, 5 days a week, for 6 weeks (30 exposures). After 65 days of recovery, rats were subjected to different neurobehavioral test to evaluate changes in performance capacity. These tests took 21 days to complete on all animals. Animals were sacrificed at the end of testing and their blood and brain (5 sections) were tested for concentrations of major neurotransmitter and metabolite.

There were no treatment-related effects on clinical signs, mortality, body weight, or on the following neurobehaviours: acoustic startle response, prepulse inhibition of acoustic startle response, total locomotor activity, tail flick response, conspecific approach, porsolt forced swim test, passive avoidance, or Morris Water Maze. In the appetitive stimulus approach sensitization test, there was an increase in the seconds to approach the novel appetitive stimulus in both JP-5 and JP-8 treated rats; however, only JP-8 treatment resulted in a significant increase. There was no difference in the amount of time spent in the unbaited stimulus areas or in total line-crossing measures. JP-5 treated rats had a significant increase in forelimb grip strength compared to the controls for all three successive trials. Serum levels of 5-hydroxyindoleacetic acid were significantly increased in JP-5 treated rats, but were significantly decreased in JP-8 treated rats. No other changes were noted in blood biochemistry. There was a significant increase in dopamine levels in the hippocampus and dihydroxyphenylacetic acid (a dopamine metabolite) in the cerebral cortex of JP-5 treated rats compared to the controls. There was a significant decrease in 5-homovanillic acid (a serotonin metabolite) in the hippocampus of JP-5 treated rats compared to the controls. JP-8 exposed rats had significantly decreased levels of dihydroxyphenylacetic acid in both the cerebellum and brainstem regions compared to the controls.

The results indicate that exposure to JP-5 or JP-8 vapour does not affect basic sensory, motor, or inhibitory functions. Nor does exposure affect the capacity of rats to learn and recall tasks of minimum complexity. Although examinations were not performed directly after the exposure period and evaluations were not conducted until at least 2 months after exposure had finished, there is no indication of any long term neurological effects in the rats. Therefore, the NOAEC is 1000 mg/m3 for JP-8 and 1200 mg/m3 for JP-5.

This study received a Klimisch score of 2 and is classified as reliable with restrictions because it is an acceptable and well-documented study report.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
1 000 mg/m³
Study duration:
subacute
Species:
rat

Effect on neurotoxicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Data on related substances have been used to 'read-across' and predict the hazard properties. A 'read-across' justification document can be found in section 13.

In this neurotoxicity study, 32 male Sprague-Dawley rats were exposed to room air, 1000 mg/m3(1 mg/L) of JP-8 jet fuel or 1200 mg/m3(1.2 mg/L) of JP-5 jet fuel 6 hours a day, 5 days a week, for 6 weeks (30 exposures). After 65 days of recovery, rats were subjected to different neurobehavioral test to evaluate changes in performance capacity. These tests took 21 days to complete on all animals. Animals were sacrificed at the end of testing and their blood and brain (5 sections) were tested for concentrations of major neurotransmitter and metabolite.

There were no treatment-related effects on clinical signs, mortality, body weight, or on the following neurobehaviours: acoustic startle response, prepulse inhibition of acoustic startle response, total locomotor activity, tail flick response, conspecific approach, porsolt forced swim test, passive avoidance, or Morris Water Maze.In the appetitive stimulus approach sensitization test, there was an increase in the seconds to approach the novel appetitive stimulus in both JP-5 and JP-8 treated rats; however, only JP-8 treatment resulted in a significant increase. There was no difference in the amount of time spent in the unbaited stimulus areas or in total line-crossing measures. JP-5 treated rats had a significant increase in forelimb grip strength compared to the controls for all three successive trials. Serum levels of 5-hydroxyindoleacetic acid were significantly increased in JP-5 treated rats, but were significantly decreased in JP-8 treated rats. No other changes were noted in blood biochemistry. There was a significant increase in dopamine levels in the hippocampus and dihydroxyphenylacetic acid (a dopamine metabolite) in the cerebral cortex of JP-5 treated rats compared to the controls. There was a significant decrease in 5-homovanillic acid (a serotonin metabolite) in the hippocampus of JP-5 treated rats compared to the controls. JP-8 exposed rats had significantly decreased levels of dihydroxyphenylacetic acid in both the cerebellum and brainstem regions compared to the controls.

The results indicate that exposure to JP-5 or JP-8 vapour does not affect basic sensory, motor, or inhibitory functions. Nor does exposure affect the capacity of rats to learn and recall tasks of minimum complexity. Although examinations were not performed directly after the exposure period and evaluations were not conducted until at least 2 months after exposure had finished, there is no indication of any long term neurological effects in the rats. Therefore, the NOAEC is 1000 mg/m3for JP-8 and 1200 mg/m3for JP-5.

Justification for classification or non-classification

No evidence of neurotoxicity to support classification

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