Isoheptane

Neurotoxicity

Description of key information

NOAEC (mouse, operant behaviour) = 1000 ppm

NOAEC (rat , neurotoxicity) = ≥ 24300 mg/m³

The weight of evidence based on an analogue approach indictaes that isoheptane in 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: acute 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: Acceptable, well documented publication/study report which meets basic scientific principles

Justification for type of information:

A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across: supporting information

Principles of method if other than guideline:

Functional observation battery (FOB) and operant behaviour testing after exposure to ISOPAR E

GLP compliance:

not specified

Limit test:

yes

Species:

mouse

Strain:

other: CFW (ChasRiver Swiss)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, MA
- Weight at study initiation: 27-40 g
- Housing: Mice were housed individually in 18x29x13 cm plastic cages containing wood chip bedding and fitted with steel wire tops.
- Diet (ad libitum): Animals used in the operant and discrimination studies were allowed to gain weight to a maximum of 35 ± 5 g by post-session feeding of 3-4 g/day of rodent chow (Rodent Laboratory Chow, Ralston-Purina, St. Louis, MO).
- Water (ad libitum)


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-24
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

inhalation: vapour

Vehicle:

unchanged (no vehicle)

Details on exposure:

Static exposure chambers were used for mice tested in FOB and ethanol discrimination protocols. Vapor generation was carried out in 29 l cylindricaljars into which liquid solvent was injected through a port onto filter paper suspended below the sealed lid. A fan then volatilized and dispersed the solvent.
Dynamic exposure chambers were used for operant conditioning and cross-dependence protocols. Operant conditioning chambers were modified for solvent vapor exposure. Vapor was generated by airflow through a bubbler in a 500 ml solvent bath. Air saturated with vapor mixed with filtered room air and was delivered to the exposure chamber.
For dependence studies, exposures were conducted in a 20.8 l rectangular tank fitted with a Teflon-lined lid into which vapors were continuously delivered in the airflow.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentrations were measured by single wavelength monitoring infrared spectrometry [Miran 1A].
Vapor concentrations were controlled by a multiple Dyna-blender interfaced with an IBM-compatible microcomputer that dictated flow rates for each test concentration.

Duration of treatment / exposure:

30 min [5 min pre-exposure to air followed by 20 min exposure to the substance (either at a single constant concentration or at increasing concentrations every five min) and 5 min post-exposure to air]

Frequency of treatment:

single exposure

Remarks:

Doses / Concentrations:
0, 2000, 4000 and 6000 ppm
Basis:
nominal conc.
functional observation battery

Remarks:

Doses / Concentrations:
0, 500, 1000, 2000, 4000, 6000 ppm
Basis:
nominal conc.
operant behaviour

No. of animals per sex per dose:

8-10 (depending on behavioral test)

Control animals:

yes, sham-exposed

Neurobehavioural examinations performed and frequency:

For the FOB studies, groups of 8 mice were each exposed to 0, 2000, 4000 or 6000 ppm ISOPAR-E for 20 minutes. During the last 2 minutes of exposure, mice were evaluated for clinical signs of toxicity and movement. Immediately after termination of exposure, mice were removed and evaluated forease of removal and handling. Righting reflex, fore- and hind-limb grip strength, landing foot splay and other behavioral parameters were also measured.

Ethanol discrimination: 9 mice/group were trained to press a lever for milk presentation. Once trained, an intraperitoneal injection of 1.25 g/kg ethanol or saline was administered, followed by further training to reinforce response to only one lever until acquisition of discrimination between 1.25 g/kg ethanol or saline in liquid was completed. Animals were then exposed to 500, 1000, 2000 or 6000 ppm ISOPAR-E for 20 min then removed and placed in behavioral test chambers.

Operant behavior: Mice were trained in two-lever operant conditioning chambers for 2 months under a fixed ratio 20 schedule of milk reinforcement.In the 1st exposure regimen, each mouse was exposed to a single concentration of 500 to 6000 ppm ISOPAR-E for 20 min, preceded and followed by 5 min air only. In the 2nd regimen, each mouse was exposed to all 5 concentrations of solvent [500, 1000, 2000, 4000, 6000 ppm] during successive 5 min periods to provide for determination of a complete concentration-effect curve within a 30 min session. This within-session method was performed in duplicate.

Cross-dependence: groups of 10 mice were continuously exposed to 2000 ppm TCE for 4 days, then TCE exposure was discontinued. Mice were evaluated for withdrawal behavior by monitoring convulsions elicited by handling, hourly for 12 hours, then at 12-24 hr intervals until recovery was complete. To assess the ability of TCE or ISOPAR-E to modify withdrawal reaction, each was administered before the 3-4 hr post-exposure assessment during the period of peak withdrawal effects [measured at 80% mice with convulsions] at concentrations of 2000 or 4000 ppm for 20 or 60 min.

Positive control:

1,1,1-trichloroethane [TCE] was tested as a positive control for comparison.

Statistics:

Concentration-effects curves for operant response rates analyzed by analysis of variance and Tukey's post-hoc comparisons. FOB used linear models approach with modification for between-subject data versus repeated measures. Continuous and count measures analyzed by means of separate general linear model procedures. Tukey's post hoc tests used to specify differences from controls.

Behaviour (functional findings):

effects observed, treatment-related

Details on results:

NEUROBEHAVIOUR:
FOB: few effects were produced for these endpoints, seen mostly at 6000 ppm. Effects included decreased arousal during the last 2 min of exposure, decreased hind-limb footsplay, increased rearing behavior at high concentrations. No effect on motor coordination was seen.

Operant conditioning: Concentration related decreases in response rates occurred in the between session protocol at 2000-6000 ppm and the within session protocol at 4000-6000 ppm.

Ethanol-like discriminative stimulus effects: Following ISOPAR-E exposure percentage of ethanol lever responses increased in a concentration-dependent manner to a maximum of 65 % at 6000 ppm. However, substitution behavior induced by ISOPAR-E for ethanol occurred at concentrations which decreased the rate of responding, indicating that responses took place at concentrations that impaired performance.

TCE cross dependence: Exposure of TCE withdrawn mice to 2000 ppm ISOPAR-E for 30-60 min or to 4000 ppm for 30 minutes caused a 40-50 % decrease in mice convulsing. At the higher concentration of 4000 ppm for 60 minutes, convulsions were suppressed by 100 %. Withdrawal reactions returned over 2 hours as ISOPAR-E suppressive effect subsided.

The narrower separation between of acute effects (6000 ppm) and lethal effect (8000 ppm) compared to TCE which showed CNS effects over a wide range of dose concentrations, indicate that the abuse levels needed to produce CNS effects would be very toxic.

Key result

Dose descriptor:

NOAEC

Remarks:

operant behaviour

Effect level:

ca. 1 000 ppm

Sex:

male

Remarks on result:

other:

Conclusions:

Exposure at 8000 ppm caused convulsions resulting in death and was not used thereafter in this study.

In this acute neurobehavioural study very few functional observational effects were seen and only at 6000 ppm. The test substance induced ethanol substitution behaviour only at 6000 ppm, a concentration that impaired performance. Operant rates were affected variously over the range of 2000 to 6000 ppm. Based on these results the NOAEC for operant behaviour was considered to be 1000 ppm.

Executive summary:

Exposure at 8000 ppm caused convulsions resulting in death and was not used thereafter in this study.

In this acute neurobehavioural study very few functional observational effects were seen and only at 6000 ppm. The test substance induced ethanol substitution behaviour only at 6000 ppm, a concentration that impaired performance. Operant rates were affected variously over the range of 2000 to 6000 ppm. Based on these results the NOAEC for operant behaviour was considered to be 1000 ppm.

Reference 2

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 NOAEL 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 NOAEL 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:

One key and two supporting read across acute neurotoxicity studies and one supporting read across sub-chronic neurotoxicity study available for assessment.

Effect on neurotoxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

There is no neurotoxicity data available for Isoheptane. However, acute neurotoxicity data is available for a structural analogue Hydrocarbons C7-C9, isoalkanes. This data is supported by sub-chronic neurotoxicity information available for another structural analogue, Light alkyl naphtha distillate and presented in the dossier. This data is read across to Isoheptane based on analogue read across and a discussion and report on the read across strategy is provided as an attachment in IUCLID Section 13.

 

Hydrocarbons, C7-C9, isoalkanes

 

An acute neurobehavioural study was reported for hydrocarbons, C7-C9, isoalkanes, in which male mice were exposed to 0, 500, 1000, 2000, 4000, or 6000 ppm in a single 20 to 30 min exposure. The study was conducted to determine if this solvent produced behavioural and pharmacologic effects similar to commonly abused inhalable solvents such as 1,1,1-trichloroethane (TCE) or ethanol. Animals were evaluated in the functional observational battery (FOB), for operant behaviour, ethanol discrimination and cross-dependence with TCE. Exposure at 8000 ppm caused convulsions resulting in death and was not used thereafter in this study. Very few FOB effects were seen and only at 6000 ppm. The test substance induced ethanol substitution behaviour only at 6000 ppm, a concentration that impaired performance. Operant rates were affected variously over the range of 2000 to 6000 ppm. Based on these results the NOAEC for operant behaviour was considered to be 1000 ppm (Balster et al., 1997).

 

In another study with hydrocarbons, C7-C9, isoalkanes, 10 male mice per dose were exposed by inhalation to 0, 1000, 2000, 4000 and 6000 ppm for 30 min, and then tested for their acute effects on locomotor activity and operant performance. The test substance produced reversible increases in locomotor activity starting at a concentration of 4000 ppm. At the same time, reversible concentration-dependent decreases in rates of schedule-controlled operant activity were observed. However, the operant performance was already impaired at 2000 ppm (Bowen and Balster, 1998). In the same publication, the results of tests perfomed with an identical study design, the test substance (under a different trade name) produced reversible increases in locomotor activity starting at a concentration of 4000 ppm. At the same time and dose, reversible concentration-dependent decreases in rates of schedule-controlled operant activity were observed. The effects caused in this case were less pronounced than those described previously (Bowen and Balster, 1998).

 

Light alkylate naphtha distillate

 

In support of the findings described above, read-across from a structurally related substance was performed. 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 (Schreiner et al., 1998).

 

Several other analogues have also been tested, namely n-heptane; n-octane; hydrocarbons, C6-C7, n-alkanes, isoalkanes, cyclics, < 5% n-hexane and alkanes, C7-10-iso- (analogue substance for iso-octane). Studies on neurotoxic effects were performed in rodents upon single and/or repeated dose inhalation exposure to the test substances. In the majority of cases, measurement of various parameters of neurobehavioral response showed minimal to no adverse effects. In some cases, however, reversible neurobehavioural effects occurred at the higher dose levels. NOAEC values for neurobehavioural effects were ≥ 1000 ppm (ca. 3500-5200 mg/m³ depending on composition), mice being much more sensitive than rats (Frantik et al., 1994; CEFIC, 2000, 2001; Lammers, 2001).

 

Therefore, Isoheptane is unlikely to present a hazard as a neurotoxicant.

 

References:

 

Frantik, E. et al. (1994). Relative Acute Neurotoxicity of Solvents: Isoeffective Air Concentrations of 48 Compounds Evaluated in Rats and Mice. Environmental

Research 66: 173-185.

 

CEFIC,(2000). The Effects of Short-term Inhalatory Exposure to n-octane on Behaviour in the Rat. Unpublished. Testing laboratory: TNO Nutrition and Food

Research Institute. Report no.: V99.429 Final. Owner company: CEFIC,. Study number: 40.144/01.04. Report date: 2000-01-12.

 

Lammers, J. H. C. M. (2001). The Effects of Short-term Inhalatory Exposure to Cypar 7 on Behaviour in the Rat. Unpublished. Testing laboratory: TNO Nutrition

and Food Research Institute. Report no.: V99.1115 Final. Owner company: CEFIC,. Study number: 40.144/01.10. Report date: 2001-02-15.

 

Balster, R. L. et al. (1997). Evaluation of the acute behavioral effects and abuse potential of a C8-C9 isoparaffin solvent. Drug and Alcohol Dependence 46:

125-135.

 

Bowen, S. E. and Balster, R. L. (1998). The Effects of Inhaled Isoparaffins on Locomotor Activity and Operant Performance in Mice. Pharmacology Biochemistry

and Behavior, 61(3): 271-280.

 

Schreiner, C. et al. (1998). Toxicity evaluation of petroleum blending streams: inhalation subchronic toxicity/neurotoxicity study of a light alkylate naphtha

distillate in rats. Journal of Toxicology and Environmental Health (Part A) 55:277-296.

 

CEFIC,(2001). The Effects of Short-term Inhalatory Exposure to Iso-octane on Behaviour in the Rat. Unpublished. Testing laboratory: TNO Nutrition and Food

Research Institute. Report no.: V99.430 Final. Owner company: CEFIC,. Study number: 40.144/01.09. Report date: 2001-02-15.