Octane

Neurotoxicity

Description of key information

NOAEC (acute/subacute, rat, visual discrimination, FOB/motor activity) > 14000 mg/m³     

NOAEC (sub-chronic, rat, neurotoxicity) >= 24300 mg/m³     

 

Based on available substance specific and read across data, Octane is unlikely to present a hazard as a neurotoxicant.

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

Reference 1

Endpoint:

neurotoxicity: short-term inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 Dec 1999 - 11 Dec 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Principles of method if other than guideline:

Effects of the test compound on cognitive performance were evaluated using a discrete-trial two-choice visual discrimination task.

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministry of Health, Welfare and Sport, State Supervisory Public Health Service, Veterinary Public Health Inspectorate, GLP Section

Limit test:

no

Species:

rat

Strain:

other: WAG/RijCrlBR

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 14 weeks
- Weight at study initiation: approximately 150 g at randomization

- Housing: From the acclimatization period onwards, the animals were housed in groups of 4 in suspended stainless steel cages until they were moved to the exposure chambers. In the exposure chambers animals were housed in groups of 4 in wire-mesh cages. Animals were acclimatized to the exposure chambers 3 days prior to the pre-exposure test week.
- Diet (ad libitum): commercial rodent diet (Rat & Mouse No. 3 Breeding Diet, RM3)
- Water (ad libitum): Tap water suitable for human consumption (quality guidelines according to Dutch legislation based on EEC Council Directive 80/778/EEC, see Annex 3) was supplied by N.V. Waterleidingbedrijf Midden-Nederland (WMN).
- Acclimation period: 10 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-24 (lowest 17.5)
- Humidity (%): 30-70 (maximum was 78)
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): artificially illuminated for 12 hours between 7.30 a.m. and 7.30 p.m.


IN-LIFE DATES: From: 1998-10-14 To: 1998-12-11

Route of administration:

inhalation: vapour

Vehicle:

other: air

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Test atmosphere was generated by pumping liquid n-octane into stainless steel tubing using peristaltic pumps. The tubing was led through a water bath at 67-68 °C and the resulting vapour was transported with an airstream from a compressed air source and added to the main airflow system.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

During exposure a total carbon analyzer was operated with 4 ports for control and 3 test concentrations. Shortly after the experiment, a stability check of the concentrations was performed.

Duration of treatment / exposure:

8 hours

Frequency of treatment:

once daily for 3 consecutive days

Remarks:

Doses / Concentrations:
0 g/m3; 1.4 g/m3 corresponding to 300 ppm; 4.2 g/m3 corresponding to 900 ppm; 14 g/m3 corresponding to 3000 ppm
Basis:
nominal conc.

No. of animals per sex per dose:

8

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for animal assignment: The rat was selected because this species is considered suitable for this type of study and was the species specified in the TNO EZ Collective project proposal. The strain of rats used in these experiments has been used extensively in behavioral studies within TNO.

Observations and clinical examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Cage side observations checked: no details given

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: clinical signs of ill health recorded prior to randomization and on Mondays during operant training: no details given

BODY WEIGHT: Yes
- Time schedule for examinations: body weight was recorded during randomization and on a weekly basis until the end of the study, and further after testing was completed on each day of exposure.

Neurobehavioural examinations performed and frequency:

LEARNING AND MEMORY TESTING: Yes
(1) Overall testing design
Visual discrimination performance: Effects of the test compound on cognitive performance were evaluated using a discrete-trial two-choice visual discrimination task.
Parameters evaluated:
General measures: trials responded to, % reinforcements obtained
Stimulus control: discrimination ratio, % ITI periods responded to
Disinhibition: repetitive errors, repetitive ITI responses
Psychomotor slowing: two choice S+ latency, short latency responses, long-latency responses, within-subject variability and single-choice SR+ latency, within-subsject variability
(2) Equipment used
- Type of equipment: The apparatus consisted of 16 operant chambers and programming and recording equipment programmed with the MedState® notation system (Med Associates, Inc., Georgia, VT). Each of the operant chambers (32x30x28 lxwxh) was equipped with two levers, two stimulus lights and a water dipper for delivering water reinforcement. A photocell assembly was mounted in the water trough in order to detect the entry of the rat´s head when obtaining the reinforcement. Each operant chamber was located in a ventilated sound-attenuated cubicle.
Although 32 rats were randomly assigned to the test groups before the start of the study, a total of 36 animals were trained.
(3) Testing and training procedures
Prior to treatment, water-deprived rats were first trained to obtain water reinforcements and to lever press using autoshaping techniques. The rats subsequently received four weeks of training on a discrete-trial light-dark visual discrimination task in order to stabilize baseline responding. Animals were trained 5 days/week, from Monday to Friday.
Test sessions consisted of 100 trials or 60 minutes whichever came first and were conducted at approximately the same time each day. On days of exposure, rats were tested immediately following the end of the exposure period. A post-exposure test was performed the day after the last exposure period in order to evaluate the persistence of effects.
Trials were signaled by the illumination of either the left or right stimulus light (S+) and the rat´s task was to depress the lever under the illuminated light in order to obtain water reward. Illumination of right and left stimulus lights was counterbalanced and occured in a predetermined semi-random order. If the rat pressed the correct lever (S+ response), the stimulus light was extinguished and a water reward (SR+) was delivered. If the initial response during a trial was on the incorrect lever (S- response), the rat was allowed to correct its mistake by pressing the lever under the illuminated stimulus light. A given trial remained in effect until the correct lever had been pressed. Trials were separated by an intertrial interval (ITI) of 10 seconds. A response during the ITI reset the ITI timer and the rat was required to wait a further 10 seconds before the initiation of the following trial.
(4) Control procedures
The correctness of the initial response on each trial was recorded.
Examination of baseline performance averaged across 5 days in the week preceding exposure (pre-day) for each rat indicated that all animals were well-trained prior to exposure.
(5) Performance measures
If the initial trial response was correct (S+ response), the latency of the lever press was also recorded (S+ response latency). If the initial response was incorrect (S- response), the number of incorrect lever responses made by the rat switching to the correct lever was recorded. During the intertrial period, lever responses were measured to determine the number of ITI periods on which 1 or more lever presses occured and the number of repetitive ITI lever responses.
From these measures, a number of dependent variables were defined to describe effects on general levels of responding, stimulus control and disinhibition, and response speed.

Statistics:

All data were analyzed using the SAS® statistical software package (release 6.12). For each test measure, probability values of p≤0.05 were consideredsignificant.
Body weights were analyzed using one-way ANOVA followed by Dunnett´s multiple comparison tests.
One-way ANOVA was conducted to examine baseline performance prior to exposure.
Treatment effects were analyzed using repeated measures analysis of variance of the data recorded during the 3-day exposure period. Huynh-Feldt adjustment of p-values of the repeated measures factor was applied in case the assumption of sphericity of observations was violated. When a significant treatment effect was demonstrated, pairwise group comparisons were performed in order to determine which solvent-treated group significantly differed from the control group. When a significant treatment-by-time interaction was demonstrated, one-way analysis of variance was performed at each test time point followed by Dunnett´s multiple comparison tests. Persistence of effects was evaluated by analysis of variance of post-exposure data.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Behaviour (functional findings):

no effects observed

Details on results:

Exposure levels used in these studies did not induce clear signs of general intoxication. No significant effects of exposure were observed on body weight.

CLINICAL SIGNS AND MORTALITY
No remarkable clinical signs were observed.

BODY WEIGHT AND WEIGHT GAIN
During the 3-day exposure period, both increases and decreases in body weight were observed in animals of all groups. However, changes in body weight were small and statistical analysis of body weight or body weight changes did not indicate any significant differences between groups.


NEUROBEHAVIOUR (Visual discrimination performance)
With respect to effects on measures of learned performance, no toxicologically relevant effects of exposure were observed. No significant effects of exposure to n-octane were observed on any measure during the 3-day exposure period. Most pronounced difference between groups was a slight increase in lever response latency in the highest exposure groups after the first 8-hour exposure period, but these group differences were not statistically significant.
During pre-week testing, pre-day testing and post-day testing a number of statistically significant differences between groups were found that were considered not to be related to exposure.

Key result

Dose descriptor:

NOAEC

Effect level:

> 14 000 mg/m³ air (nominal)

Sex:

male

Basis for effect level:

other: overall effects (no effects) highest dose tested

Remarks on result:

other:

Conclusions:

In conclusion, short-term high-level exposure to n-octane did not induce any toxicologically significant effects on measures of learned performance.

Executive summary:

In conclusion, short-term high-level exposure to n-octane did not induce any toxicologically significant effects on measures of learned performance.

Reference 2

Endpoint:

neurotoxicity: inhalation

Remarks:

other: acute and subacute

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 Dec 1999 - 11 Dec 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Principles of method if other than guideline:

Neurobehavioral functioning was evaluated using selected measures from a standardized functional observational battery (FOB) and motor activity assessment protocol similar to that used in the WHO/IPCS Collaborative Study on Neurotoxicity Assessment (Moser and MacPhail, 1992; Moser et al., 1997a; Moser et al., 1997b).

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministry of Health, Welfare and Sport, State Supervisory Public Health Service, Veterinary Public Health Inspectorate, GLP Section

Limit test:

no

Species:

rat

Strain:

other: WAG/RijCrlBR

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 14 weeks
- Weight at study initiation: approximately 250 g at randomization

- Housing: Animals were housed in groups of 5 in suspended stainless steel cages under conventional conditions. After randomization animals were moved to the exposure chambers and housed individually in wire-mesh cages.
- Diet (ad libitum): commercial rodent diet (Rat & Mouse No. 3 Breeding Diet, RM3)
- Water (ad libitum): Tap water suitable for human consumption (quality guidelines according to Dutch legislation based on EEC Council Directive 80/778/EEC, see Annex 3) was supplied by N.V. Waterleidingbedrijf Midden-Nederland (WMN).
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-24 (lowest 17.5)
- Humidity (%): 30-70 (maximum was 78)
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): artificially illuminated for 12 hours between 7.30 a.m. and 7.30 p.m.


IN-LIFE DATES: From: 1998-11-18 To: 1998-12-11

Route of administration:

inhalation: vapour

Vehicle:

other: air

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Test atmosphere was generated by pumping liquid n-octane into stainless steel tubing using peristaltic pumps. The tubing was led through a water bath at 67-68 °C and the resulting vapour was transported with an airstream from a compressed air source and added to the main airflow system.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

During exposure a total carbon analyzer was operated with 4 ports for control and 3 test concentrations. Shortly after the experiment, a stability check of the concentrations was performed.

Duration of treatment / exposure:

8 hours

Frequency of treatment:

single exposure and once daily for 3 consecutive days

Remarks:

Doses / Concentrations:
0 g/m3; 1.4 g/m3 corresponding to 300 ppm; 4.2 g/m3 corresponding to 900 ppm; 14 g/m3 corresponding to 3000 ppm
Basis:
nominal conc.

No. of animals per sex per dose:

8

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for animal assignment: The rat was selected because this species is considered suitable for this type of study and was the species specified in the TNO EZ Collective project proposal. The strain of rats used in these experiments has been used extensively in behavioral studies within TNO.

Observations and clinical examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Cage side observations checked: no details given

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to randomization (no details given)


BODY WEIGHT: Yes
- Time schedule for examinations: body weight was recorded during randomization and on days of testing

Neurobehavioural examinations performed and frequency:

FUNCTIONAL OBSERVATIONAL BATTERY: Yes
- Parameters checked:
Neuromuscular: gait, forelimb and hindlimb gripstrength, landing foot splay
Sensorimotor: response to tail pinch, click, touch, approach of a visual object
Convulsive: clonic and tonic movements
Excitability: arousal
Activity: motor activity

- Minimization of bias: Technicians were blind to treatment status of animals: Yes
- Site of testing: open arena (77x55x7 cm)
- Time schedule for examinations: FOBs were carried out 6 days prior to the start of exposure and immediately following the first and third exposure period.
- Duration of observation period for open field observations: 1 minute


LOCOMOTOR ACTIVITY: Yes
- Type of equipment used: automated quantitative microprocessor-based video image analysis system (Ethovision, Noldus Information Technology b.v., The Netherlands)
- Length of session, number and length of subsessions: 30 minutes
- Parameters measured: The position of the rat was continuously monitored throughout the test session concerning distance traversed, number of movements and mean velocity. Spontaneous motor activity was expressed as the total distance run in a test period. In addition, quantitative measures of locomotor speed and patterns of locomotor activity were also recorded.

Statistics:

All data were analyzed using the SAS® statistical software package (release 6.12). For each test measure, probability values of p≤0.05 were consideredsignificant.
Continuous variables from the FOB were analyzed using ANOVA. Treatment effects were analyzed using repeated measures analysis of variance. Group comparisons were made using Dunnett´s multiple comparison tests. Rank data were analyzed by Kruskal-Wallis one-way analysis of variance.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Behaviour (functional findings):

no effects observed

Details on results:

Exposure levels (single exposure and once daily for 3 consecutive days) used in these studies did not induce clear signs of general intoxication. No significant effects of exposure were observed on body weight or on body temperature.

CLINICAL SIGNS AND MORTALITY
No remarkable clinical signs were observed.

BODY WEIGHT AND WEIGHT GAIN
During the 3-day exposure period, mean body weights were only slightly changed. A small decrease was observed in the 14 g/m3 group during the exposure period. Repeated measures analysis of variance of body weight data indicated a significant treatment-by-time interaction. However, analysis of variance of body weight data at each test time point indicated that there were no significant differences between groups.


NEUROBEHAVIOUR
Statistical analysis of functional observations and motor activity data indicated group differences in landing foot splay, hindlimb gripstrength. Significant interaction effects (distance traversed, number of movements, mean velocity) were observed for measures of motor activity.
However, none of the exposed groups appeared to be significantly different from the control group for any of the above measures.
FOB: Following exposure, no changes in functional observational measures were observed that could be related to exposure to n-octane.
Motor activity: Subsequent statistical analysis of motor activity data at each test time point did not indicate any of the exposure groups to be significantly different from the control group.

Key result

Dose descriptor:

NOAEC

Effect level:

> 14 000 mg/m³ air (nominal)

Sex:

male

Basis for effect level:

other: overall effects (no effects) highest dose tested

Remarks on result:

other:

Conclusions:

In conclusion, short-term high-level exposure to n-octane did not induce any toxicologically significant effects on functional observations and measures of motor activity.

Executive summary:

In conclusion, short-term high-level exposure to n-octane did not induce any toxicologically significant effects on functional observations and measures of motor activity.

Reference 3

Endpoint:

neurotoxicity: sub-chronic inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-guideline study.

Justification for type of information:

The justification for read across is provided as an attachment in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

equivalent or similar to guideline

Guideline:

other: (OECD) Guideline 413 for Subchronic Inhalation Toxicity (1981)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Male and nulliparous, nonpregnant female Sprague-Dawley rats (VAF/ Plus Crl:CD BR)
- Source: Charles River Laboratories, Kingston, NY
- Age at study initiation: approximately 5 wk old when purchased, acclimated for approximately 2 wk prior to the initiation of the study
- Housing: Animals were housed individually in suspended stainless steel wire mesh cages in air-conditioned rooms
- Diet (e.g. ad libitum): Certified rodent diet 5002 (PMI Feeds, Inc., St. Louis, MO) ad libitum
- Water (e.g. ad libitum): water from an automated watering system was available ad libitum
- Acclimation period: 2 weeks. All animals were assigned a temporary identification number at receipt and examined by the staff veterinarian during the acclimation period.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26°C
- Humidity (%): 40-70% relative humidity
- Photoperiod (hrs dark / hrs light): 12/12 during the acclimation and all nonexposure periods

Route of administration:

inhalation: vapour

Vehicle:

other: nitrogen

Details on exposure:

Rats were exposed to wholly vaporized LAND-2 generated in nitrogen, by inhalation in whole-body exposure cages 6 h/d, 5 d/wk for 13 wk at analytical concentrations of 668, 2220, and 6646 ppm (2.4, 8.1, and 24.3 g/m3). Exposure levels were determined three times daily by gas chromatography. The highest concentration was approximately 75% of the lower explosive limit. Animals’ positions in the cages were rotated for each exposure to ensure uniform exposure of every animal.

Chamber Operation: Animals were housed individually in wire mesh, stainless steel cages within a 1000-L glass and stainless steel exposure chamber. Chamber temperature and humidity were monitored every half hour during exposure and maintained, to the extent possible, within the ranges of 20–24°C temperature and 40–60% relative humidity. Animals did not receive food or water during the exposure period. Exposure chambers were operated dynamically at a calibrated airflow rate of 200 L/min (lpm). Recordings of airflow and static pressure were made every half hour. All animals remained in the chamber for a minimum of 30 min at the end of exposure while the chamber was operated using clean air only. Chambers were exhausted through a system of coarse filter, HEPA filter, and charcoal filter.

Atmosphere Generation: LAND-2 was pumped directly from the 5-gal container, housed within a freezer constantly flushed with nitrogen, using a laboratory pump, equipped with a piston, that was insulated within a styrofoam container. An ice bag was placed on top of the piston to keep the piston chamber cold to inhibit volatilization of LAND-2 in the pump and delivery lines. LAND-2 was delivered onto the central glass helix of a countercurrent volatilization chamber (one generator per chamber). The glass helix was heated by an internal nichrome wire inserted in the center of the glass tube that supported the helix (external to the volatilization chamber) and was controlled by a variable autotransformer. House-line nitrogen delivered from a regulator with a backpressure gauge was divided with a stainless steel T into the generation flow system and a purge flow system. Purge nitrogen was delivered to the bottom of the tube containing the nichrome wire to continuously purge the area surrounding the wire, protecting it from oxidation. Nitrogen for the generation system was directed through a flowmeter to the ball-and-socket joint at the bottom of the volatilization chamber, flowed up the chamber, passed over the coil, and volatilized the test material. The LAND-2-laden nitrogen flowed through a T tube at the top of the volatilization chamber into the turret of a 1-m3 glass and stainless steel exposure chamber, where it mixed with room air to appropriate exposure concentrations as it was drawn into the chamber (flow rate of 200 lpm). Control animals were sham-exposed to nitrogen alone introduced into the turret and mixed with air in the chamber.

Exposure Chamber Monitoring: Samples for determination of analytical exposure levels and the major components of LAND-2 vapor were withdrawn by vacuum pump from the breathing zone in the exposure chambers three times per exposure for treated groups and once per exposure for controls. Samples were pulled through Teflon lines into the multipositional control module and directed to a Hewlett Packard 5890II gas chromatograph, equipped with a flame ionization detector for analysis by ASTM method D5134-92 (ASTM, 1992). Composition and stability of the test material were evaluated by characterizing neat LAND-2 and comparing the major components in the neat and generated atmospheres at the beginning and end of the study. Particle size distribution measurements of any background aerosol were performed once during each exposure for chambers and room air using a TSI Aerodynamic Particle Sizer. Samples were drawn for 20 s at a rate of 5 L/min. Mean mass aerodynamic diameter (MMAD), geometric standard deviation (GSD), and total mass concentration (TMC) were calculated. Nominal concentrations (mg/m3) were calculated from the loss of weight from the generation apparatus divided by the total air flow through the chamber during exposure. This value was converted to parts per million (ppm) using an average molecular weight for this mixture of 89.2.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Determined three times daily by gas chromatography.
Composition and Uniformity Chamber Gas Chromatographic Results (% Weight): n-Butane: 3.21; iso-Pentane: 34.343; 2,3-Dimethylbutane: 12.977, 2-Methylpentane: 4.096; 2,4-Dimethylpentane: 5.663; 2,3-Dimethylpentane: 2.680; 2,2,4-Trimethylpentane: 16.885; 2,3,4-Trimethylpentane: 3.578; 2,3,3 –Trimethylpentane: 4.505; 2,2,5-Trimethylhexane: 2.499.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 hours/day, 5 days/week

Remarks:

Doses / Concentrations:
0, 668, 2220, and 6646 ppm (0, 2.4, 8.1, and 24.3 g/m3)
Basis:
analytical conc.

No. of animals per sex per dose:

12 animals/sex/group

Control animals:

yes, concurrent no treatment

Details on study design:

Neurobehavioral evaluations of motor activity (MA) and functional operational battery (FOB) were performed pretest and during wk 5, 9, 14, and 1 8 (recovery groups). Animals were not exposed to LAND-2 on the days of neurobehavioral testing. Exposure days were added to ensure that each animal received at least 65 exposures.

Following 13 wk of exposure, 12 animals/sex/group were necropsied and microscopic examination was performed on selected tissues. Nervous tissue from 6 rats/sex/group was also examined microscopically. At the end of the 4-wk recovery period, 12 animals/sex from the high and control groups were necropsied and selected tissues examined microscopically.

Observations and clinical examinations performed and frequency:

Animals were evaluated twice daily for mortality and gross signs of toxicological or pharmacological effects. During each exposure, rats were observed as a group once for abnormal behavior. Detailed physical examinations were performed twice pretest and weekly during the study. Ophthalmoscopic evaluations were performed pretest and just prior to the scheduled sacrifices at wk 14 (terminal) and 18 (recovery).

Body Weights and Food Consumption. All animals were weighed twice pretest, weekly during the study period, and prior to scheduled sacrifice. Food consumption was measured once during the week prior to treatment initiation and over a 6-d interval each week during the study period.

Specific biochemical examinations:

Hematology and Clinical Chemistry. Blood was obtained by venipuncture of the orbital sinus from 12 fasted rats/sex/group under light carbon dioxide/oxygen anesthesia at terminal sacrifice (wk 14) and at the end of the recovery period (wk 1 8). Hematology parameters measured were hemoglobin concentration, hematocrit, erythrocyte and platelet counts, mean corpuscular volume, mean corpuscular hemoglobin and hemoglobin concentration, prothrombin time, activated partial thromboplastin time, total and differential leukocyte counts, erythrocyte morphology, and reticulocyte count. Clinical chemistry parameters were aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, blood urea nitrogen, fasting glucose, total protein, albumin, globulin (calculated), albumin/globulin ratio (calculated), creatinine, total bilirubin, sodium, potassium, chloride, calcium, and inorganic phosphorus.

Neurobehavioural examinations performed and frequency:

Animals were transported from the room in which they were housed during non-exposure hours to the neurotoxicity laboratories. Behaviors were evaluated pretest and during wk 5, 9, 14, and 18 (recovery groups). Temperature, humidity, and illumination were measured and recorded to minimize variation in environmental conditions during evaluations. Noise levels were not recorded. Testing was staggered over 8 sessions within a 4-d period, each session consisting of approximately 2 rats/sex/treatment group.

Motor Activity. Locomotor activity was monitored as a function of the number of beam breaks in an activity box, using an automated photo- beam activity system. Sessions were 60 mm in length divided into twelve 5-mm intervals. Rats were evaluated at pretest, at 3 time points during the treatment period (wk 5, 9, and 1 4), and at the end of the recovery period. Treatment groups were counter balanced across test times. Three sets of analyses were performed. The first analysis was conducted using the pre-dose data with animals nested within the interaction effect of sex crossed with treatment group, with a profile measure across the 12 time intervals. The second analysis used data from the 3 treatment time points, and animals were nested within the interaction effect of sex crossed with treatment group, with a profile measure across the 12 time intervals nested within the 3 time periods. The third analysis was similar to the first, using the recovery data. All three analyses tested for treatment effects, sex differences, treatment group by sex interactions, and these effects crossed with periods (second analysis only) and intervals. Analyses were repeated using transformed rank data to achieve a normal distribution of the residuals. Residuals from the models were tested for normality by the Shapiro-Wilk W-test or the Kolomogorov D-test.

Functional Operational Battery. The battery was comprised of home- cage evaluations (posture, vocalizations, and palpebral closure), handling evaluations (reactivity to general stimuli, signs of autonomic function), open-field behavior (arousal level and gait, urination and defecation frequency, convulsions, tremor, abnormal behaviors, piloerection, and exophthalmos), and reflex assessments (response to visual and auditory stimuli, tail pinch, pupillary function). Animals were also evaluated for forelimb and hindlimb grip strength, landing foot splay, and air righting ability. For landing foot splay, a small dot of paint was applied to each hindpaw. The rat was dropped from a height of 2 ft above a flat surface and the distance between the marks left by the hindpaws was measured in centimeters. To evaluate air righting reflex ability, the rat was held upside down, dropped from a height of 2 ft above a container of bedding, and the landing position was observed. Treatment groups were counterbalanced across test times. The observer performing the evaluation did not know the identity of the animal’s dose group.

Sacrifice and (histo)pathology:

All animals were sacrificed by intraperitoneal injection of sodium pentobarbital, and tissues were preserved in situ by transcardial perfusion with phosphate-buffered saline (pH 7.4) followed by 4% paraformaldehyde/1 % glutaraldehyde in the same buffer. Animals were killed at termination of 13 wk of exposure (week 13 terminal sacrifices performed during week 14) or at the completion of the 4-wk recovery period (control and high-dose groups only). A complete macroscopic examination was performed on all animals and 12 organs were weighed: adrenals, brain, heart, kidneys, liver, lung, ovaries, prostate, spleen, testes (with epididymides), thymus, and uterus. The length and width of the brain of each rat was measured.

Thirty-nine tissues were preserved from all animals in all dose groups. Tissues from all animals in the control and high-dose groups were processed, embedded in paraffin, mounted on glass slides, and stained with hematoxylin and eosin for histopathological examination. The kidneys of selected animals were also stained with Mallory-Heidenhain stain. In addition, tissues of the nervous system were fixed for all animals. Brain, spinal cord, ganglia, and spinal nerve roots were processed, embedded in paraffin, mounted on glass slides, and stained with hematoxylin-eosin, Luxol fast blue, and Sevier-Munger stains. Peripheral nerve sections (sciatic, tibial, sural, and optic) were embedded in glycol methacrylate and stained with toluidine blue. Slides of nervous system tissues were examined from animals (6/sex/group) designated through random selection for neuropathology, in the control and high-dose groups sacrificed at the end of 13 weeks of exposure. Specific brain regions examined were forebrain, cerebral cortex, hippocampus, basal ganglia, midbrain cerebellum and pons, and medulla.

Statistics:

Statistical evaluations were performed on the following parameters: body weights, body weight change from wk 0, and food consumption; hematology and clinical chemistry; and organ weights, organ/terminal body weight ratio, and organ/brain weight ratio. Barlett’s test at 1 % significance, two-sided risk level, was used to determine if groups had equal variance. All other tests were conducted at 5% and 1 % significance, two- sided risk level. Parametric procedures were standard one-way analysis of variance (ANOVA) using F distribution for significance. If significant differences among means were indicated, Dunnett’s test was used to determine significant differences from controls. The Kruskal-Wallis test was the nonparametric procedure for testing equality of means, and if differences were indicated, Dunn’s summed rank test was used to determine differences from controls.

A statistical test for trend in the dose levels was also performed, using standard regression techniques with a test for trend and lack of fit where variances were equal orJonckheere’s test for monotonic trend in nonparametric cases.

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 examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Behaviour (functional findings):

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Other effects:

not examined

Description (incidence and severity):

Migrated information from 'Further observations for developmental neurotoxicity study'



Details on results (for developmental neurotoxicity):not applicable (migrated information)

Details on results:

All animals survived the treatment period and were sacrificed according to study design at the end of 13 wk or at 18 wk (recovery groups). No test-related observations were noted in the exposure chambers during any exposure period for any treatment groups or during non-exposure periods. From weekly clinical observations, the only apparent treatment-related finding was an increased incidence of red facial staining in both male and female rats in the high dose group. No LAND-2-related ocular disease was observed. All groups showed similar mean body weights, body weight gains, and food consumption.

At wk 13 terminal sacrifice, there was a statistically significant decrease, relative to control values, in hemoglobin (5%), hematocrit (5%), and erythrocytes (7%) in blood of high dose males (data not shown). The hemoglobin was still decreased (4%) after the 28-d recovery period. However, because these differences were small and within the historical range for control animals in this laboratory, they are not considered toxicologically relevant. Clinical chemistry results showed a statistically significant decrease in aspartate aminotransferase (AST 32%) and alanine aminotransferase (ALT 46%) in females of the high-dose group (data not shown) compared to controls. However, several control female rats had elevated AST and ALT relative to the other nine female rats in the group and historical controls. Comparison of ALT and AST values from high-dose females with these elevated concurrent control values produced a statistical significance that is not toxicologically relevant. These results are not considered LAND-2 related.

NEUROBEHAVIORAL STUDIES

Motor Activity. Shapiro-Wilk analysis of data from the predose period indicated that the only statistically significant effects on response pattern occurred in the low-dose group due to inactivity among females for intervals 6—8, and increased activity of males during interval 10 (data not shown). Data from the treatment intervals and the recovery period were analyzed based on the Blom transformed data because the residuals from the model were not normally distributed by the Shapiro—Wilk statistic at the .01 level. There were statistically significant differences in the number (ct < .04) and relative pattern (cx. < .02) of beam breaks among the dose groups over the treatment testing periods. There were expected differences between sexes, and pattern differences across the 1 2 measuring intervals. In the recovery period in which only the room air controls and the high-dose animals were evaluated, there were no dose-group-related differences in response. Overall, dose-group differences did not occur in a dose-related pattern. Although statistically significant, the magnitudes of the differences were not large, and none of the treatment-group differences was larger than differences seen during the pre-dose period.

Functional Operational Battery. No differences were detected in the distance between foot splay for male or female rats in any dose group over any time interval evaluated. Grip strength of both fore- and hindlimbs in general increased from pretest through wk 14 for both sexes in all treatment groups. Values for control and high-dose recovery animals were lower at wk 18 than in previous treatment weeks but similar between the groups. There was no test-material-related effect on any endpoint evaluated within the functional operational battery of tests.

Pathology. At the wk 13 terminal sacrifice there were statistically significant dose-related increases in absolute and relative kidney weights in males of all 3 treatment groups. The kidney weights of high-dose males remained elevated after the recovery period. These increases correlated with microscopic observations of hyaline droplet formation in the proximal convoluted tubules considered to contain an alpha2-microglobulin-hydrocarbon complex, based on positive staining reaction by the Mallory-Heidenhain method, and increase in incidence and severity of nephropathy and dilated tubules at the corticomedullary junction. These microscopic finding are characteristic of ‘light hydrocarbon nephropathy” also known as hyaline droplet nephropathy and are male rat specific and are not considered relevant to humans. Statistically significant increases in absolute and relative liver weights were observed in high-dose male and female rats at wk 13 sacrifice. Differences were not present after the recovery period and had no microscopic correlate. Lung and brain weights were comparable to controls. Lungs were macroscopically and microscopically comparable to controls. Brain length and width measurements showed no test-material-related effects. There were no microscopic findings in the brain, spinal cord, or peripheral nerves that could be attributable to exposure to LAND-2.

Dose descriptor:

NOAEC

Remarks:

Neurotoxicity

Effect level:

> 6 646 ppm (analytical)

Sex:

male/female

Basis for effect level:

other: converted to 24.3 g/m3

Remarks on result:

other:

Dose descriptor:

NOAEC

Remarks:

subchronic toxicity

Effect level:

2 220 ppm (analytical)

Sex:

male/female

Basis for effect level:

other: converted to 8.1 g/m3

Remarks on result:

other:

Rats were exposed to wholly vaporized LAND-2 generated in nitrogen, by inhalation in whole-body exposure cages 6 h/d, 5 d/wk for 13 wk at analytical concentrations of 668, 2220, and 6646 ppm (2.4, 8.1, and 24.3 g/m3). Neurobehavioral evaluations of motor activity (MA) and functional operational battery (FOB) were performed pretest and during wk 5, 9, 14, and 1 8 (recovery groups). Animals were not exposed to LAND-2 on the days of neurobehavioral testing. Exposure days were added to ensure that each animal received at least 65 exposures. Following 13 wk of exposure, 12 animals/sex/group were necropsied and microscopic examination was performed on selected tissues. Nervous tissue from 6 rats/sex/group was also examined microscopically. At the end of the 4-wk recovery period, 12 animals/sex from the high and control groups were necropsied and selected tissues examined microscopically.The NOAEC of LAND-2 for subchronic toxicity is 2220 ppm and >6646 ppm for neurotoxicity.

Conclusions:

The NOAEC of LAND-2 for subchronic toxicity is 2220 ppm corresponding to 24.3 g/m3 and >6646 ppm corresponding to 8.1 g/m3 for neurotoxicity.

Executive summary:

The NOAEC of LAND-2 for subchronic toxicity is 2220 ppm corresponding to 24.3 g/m3 and > 6646 ppm corresponding to 8.1 g/m3 for neurotoxicity.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

24 300 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

2 key substance specific short-term studies available and 1 key sub-chronic study available from a structural analogue

Effect on neurotoxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Short-term neurotoxicity data is available for Octane. Additionally, sub-chronic data is available for structural analogue, light alkylate naphtha distillate. This data is read across to based on analogue read across and a discussion and report on the read across strategy is provided as an attachment in IUCLID Section 13.

 

Octane

In a short-term neurotoxicity study (CEFIC, 2000), groups of male rats (8/dose level) were exposed by inhalation to 0, 1400, 4200 or 14000 mg/m³ (corresponding to ca. 300, 900 and 3000 ppm) octane, 8 h per day, for 3 consecutive days. Animals were tested daily for effects on motor activity, functional observation measures and learned performance of a visual discrimination task. No clear signs of general intoxication and no significant effects on any of these parameters were observed up to and including the highest dose, therefore the NOAEC was considered to be greater than 14000 mg/m³.

 

Light alkylate naphtha distillate

In a key study (Schreineret al., 1998), rats (12/sex/group) were exposed by inhalation to a light alkylate naphtha distillate ( LAND-2, C4–C10; CAS No. 64741-66-8) at 0, 668, 2220 or 6646 ppm, 6 h per day, 5 days per week, for 13 weeks; 12 additional rats per sex in the control and high dose groups were selected for a 4-week recovery period after the final exposure. Besides standard parameters of subchronic toxicity, neurotoxicity evaluations were conducted and consisted of motor activity and a functional operational battery (FOB) measured pretest, during weeks 5, 9, and 14 of the study, and after the 4-week recovery period. No exposure-related mortality or signs of general intoxication were observed. Significant increases both in absolute and relative kidney weights were noted in males at the highest dose and correlated with hyaline droplet formation and renal nephropathy observed microscopically. These effects in male rats, however, were considered to be of no toxicological significance for humans. In both sexes, liver weights were increased at the highest dose, but no correlation was seen in microscopic examinations. Moreover, the effect appeared to reversible after the 4-week recovery period. Exposure to LAND-2 did not result in neurotoxicity as assessed by motor activity measurements, FOB, or neuropathology. The no-observed-effects level (NOAEC) for LAND-2 was 2220 ppm (corresponding to ca. 8100 mg/m³) for subchronic toxicity and ≥ 6646 ppm (corresponding to 24300 mg/m³) for neurotoxicity.

Justification for classification or non-classification

Based on available data from Octane and a structural analogue, Octane is unlikely to present a hazard as a neurotoxicant.