Isooctane

Neurotoxicity

Description of key information

NOAEC (rat) > 14000 mg/m³

 

Based on available read across data, isooctane 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: inhalation

Remarks:

other: acute and subacute

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 Jan 1999 - 12 Feb 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

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:

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:

Inspectorate for Health Protection, Commodities and Veterinary Public Health, Ministry of Health, Welfare and Sport

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: Prior to randomization, the animals were housed individually in macrolon Type III cages (Tecniplast Type 2154F) with wood-shavings on the floor. After randomization animals were 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: 9 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 and in exposure chambers: 19-24 (lowest 18.5)
- Humidity (%): 31-70 (exposure chamber: 30-55)
- 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 until 1999-01-13. From 1999-01-13 onwards lights were on from 07:00 a.m. to 07:00 p.m.


IN-LIFE DATES: From: 1999-01-20 To: 1999-02-12

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 60 °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. In addition, the test atmosphere of the low concentration port was continuously analysed during exposure by a separate total carbon analyzer which was used to stabilize the level of the concentration. 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 total distance run, 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:

effects observed, treatment-related

Behaviour (functional findings):

no effects observed

Details on results:

Exposure levels (single exposure and once daily for 3 consecutive days) used in these studies were sufficiently high to induce signs of general intoxication. Signs of intoxication, however, were very mild.

CLINICAL SIGNS AND MORTALITY
No remarkable clinical signs were observed.

BODY WEIGHT AND WEIGHT GAIN
During the 3-day exposure period, mean body weights decreased in the 14 g iso-octane/m3 group. Analysis of covariance at each test time point revealed a significant effect of exposure after the first and the third 8-hour exposure period. Post-hoc group comparisons showed that mean body weight in the 14 g iso-octane/m3 group was significantly decreased when compared to the control group at both test time points.


NEUROBEHAVIOUR
FOB: Following exposure, no changes in functional observational measures were observed that could be related to exposure to iso-octane.
Motor activity: Statistical analysis of motor activity data at each test time point did not indicate any of the exposure groups to be significantly differentfrom 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 2,4-dimethylhexane 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 2,4 -dimethylhexane did not induce any toxicologically significant effects on functional observations and measures of motor activity.

Reference 2

Endpoint:

neurotoxicity: short-term inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 Dec 1998 - 12 Feb 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

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:

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:

Inspectorate for Health Protection, Commodities and Veterinary Public Health, Ministry of Health, Welfare and Sport

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 200 g at randomization


- Housing: From the acclimatization period onwards, the animals were housed in groups of 4 in macrolon Type III cages (Tecniplast Type 2154F) with wood shavings on the floor. From 5 weeks prior to the pre-exposure test week until the end of the experiment the animals were housed in groups of 4 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: 12 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 and in exposure chambers: 19-24 (lowest 18.5)
- Humidity (%): 31-70 (exposure chamber: 30-55)
- 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 until 1999-01-13. From 1999-01-13 onwards lights were on from 07:00 a.m. to 07:00 p.m.


IN-LIFE DATES: From: 1998-12-16 To: 1999-02-12

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 60 °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. In addition, the test atmosphere of the low concentration port was continuously analyzed during exposure by a separate total carbon analyzer which was used to stabilize the level of the concentration. 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-subject 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 after the first and the third exposure period. 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
Body weights were not decreased more in iso-octane-exposed animals compared to controls.


NEUROBEHAVIOUR (Visual discrimination performance)
Visual discrimination performance was also not significantly changed after exposure to iso-octane. A number of statistically significant effects were observed during testing prior to exposure and during the exposure period, but no significant differences between iso-octane-exposed groups and the control group were observed for general measures of responding, measures of stimulus control, measures of disinhibition or measures of psychomotor speed.

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 2,4-dimethylhexane did not induce any toxicologically significant effects on measures of learned performance.

Executive summary:

In conclusion, short-term high-level exposure to 2,4 -dimethylhexane did not induce any toxicologically significant effects on measures of learned performance.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

14 000 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

Two key and one supporting read across study from structural analogues are 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 Isooctane. However, neurotoxicity data is available for structural analogues, 2,4-dimethylhexane and light alkylate naphtha distillate and presented in the dossier. 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.

 

2,4-dimethylhexane

In a key study (CEFIC, 2001), 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) of 2,4-dimethylhexane (Alkanes, C7-10-iso-, CAS No. 90622-56-3), 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. The exposure levels used were sufficiently high to induce very mild signs of general toxicity (slightly decreased body weights). No significant neurobehavioural effects were observed up to the highest dose, therefore the NOAEC was considered to be greater than 14000 mg/m³ (CEFIC, 2001).

 

Light alkylate naphtha distillate

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 hydrocarbons, C7 -C9, isoalkanes. 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; Lammers, 2001; Balster et al., 1997; Bowen and Balster, 1997; Schreiner et al. 1998).

 

Therefore, Isooctane 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.

Justification for classification or non-classification

Based on the available read across data from structural analogues, Isooctane does not warrant classification as a neurotoxicant under the new Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP).