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Neurotoxicity

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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 indicates that hydrocarbons, C7-C9, isoalkanes are unlikely to present a hazard as neurotoxicant.

Key value for chemical safety assessment

Effect on neurotoxicity: via inhalation route

Link to relevant study records
Reference
Endpoint:
neurotoxicity: acute inhalation
Type of information:
experimental study
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
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 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.

Additional information

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).

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, the hydrocarbons, C7-C9 aliphatics are unlikely to present a hazard as neurotoxicants.

 

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.

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.

Justification for classification or non-classification