Methylcyclopentane

Neurotoxicity

Description of key information

There is no data available for Methylcyclopentane. Data available from a structural analogues, n-Hexane and 2-methylpentane, are used as read across.

One read across key neurotoxicity study of commercial hexane was found (API, 1990e; Klimisch = 2). Results showed no effects to behavior or evidence of toxicity.

Key value for chemical safety assessment

Effect on neurotoxicity: via oral route

Endpoint conclusion

Endpoint conclusion:

no study available

Effect on neurotoxicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

neurotoxicity: sub-chronic inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1989-04-03 to 1989-07-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study is classified as reliable without restrictions because it is well documented and follows OECD Guideline 424.

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 424 (Neurotoxicity Study in Rodents)

Deviations:

yes

Remarks:

On a single occasion a control group female and high exposure female were mis-dosed. Due to it being a single instance, it is not considered to have affected the outcome of the study.

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Canada, Inc.
- Age at study initiation: 7-8 weeks of age
- Weight at study initiation: 233-271 g male, 185-239 g female
- Housing: individually in stainless steel wire mesh cages, identified by ear notching
- Diet (e.g. ad libitum): Purina Rodent Laboratory Chow, ad libitum
- Water (e.g. ad libitum): reverse osmosis sterilized water, ad libitum
- Acclimation period: 2 weeks male, 3 weeks female


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 25-79
- Photoperiod (hrs dark / hrs light): 12 hrs light/12 hrs dark


IN-LIFE DATES: From: April 3, 1989 To: July 14, 1989

Route of administration:

inhalation: vapour

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: cylindrical stainless steel and glass whole-body chambers, 410 l
- Method of holding animals in test chamber: cages
- Method of conditioning air: Test substance was metered into a glass column that was wrapped with a heating element and filled with glass beads. Dry compressed air moved through the column where it mixed with the test substance before entering the exposure chamber.
- Temperature, humidity, pressure in air chamber: 20-24 degree C, 30-70% humidity,
- Air change rate: 12-15 per hour


TEST ATMOSPHERE
- Brief description of analytical method used: pre-study, samples were taken from sampling points in the breathing zone and analyzed using GC. Samples were also analyzed with GC daily during the first exposure week, and weekly thereafter. During exposure, samples were taken every 30 min. and analyzed using an infrared gas analyzer.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

896 ppm
2,996 ppm
9,006 ppm

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 hrs/day, 5 days/week

Remarks:

Doses / Concentrations:
0, 900, 3000, 9000 ppm
Basis:
nominal conc.

No. of animals per sex per dose:

12 male, 12 female

Control animals:

yes, sham-exposed

Observations and clinical examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: mortality and clinical signs

BODY WEIGHT: Yes
- Time schedule for examinations: weekly, on behavioral testing days

FOOD CONSUMPTION: Yes
Weekly

Neurobehavioural examinations performed and frequency:

FUNCTIONAL OBSERVATIONAL BATTERY: Yes
- Parameters checked: body position, locomotor activity, bizarre behavior, tremors, piloerection, respiratory rate, defecation, vocalization, pupil size, lacrimation, salivation, urinary staining, diarrhea, body tone, abdominal tone, pinna reflex, extensor thrust, tail pinch, toe pinch, auricular startle, gait, limb rotation, visual placing, air righting reflex, pupillary reflex, positional passivity, urination, grip strength, hindlimb splay,
- Description of procedures:
- Minimization of bias:
- Technicians were blind to treatment status of animals: Yes
- Site of testing: 2' square of plexiglass
- Time schedule for examinations: day 0, 1, 7, 14, 35, 63, and 91
- Description of equipment where required: Both hindlimb and forelimb grip strength was tested using a Chatilon strain gauge.

LOCOMOTOR ACTIVITY: Yes
- Type of equipment used: figure 8 activity monitor
- Length of session, number and length of subsessions: 1 hr, 6 10 min sessions
- Time schedule for examinations: pre-study and days 34, 62, and 90

Sacrifice and (histo)pathology:

- Number of animals sacrificed: 8 per group
- Parameters measured:
- Brain weight: yes
- Length and width of brain: yes
- Procedures for perfusion: Ringer's solution with heparin and sodium nitrate, then gluteraldehyde, formaldehyde, calcium chloride, picric acid, and cacodylate buffer.
- Number of animals perfused: 8
- Tissues evaluated: peripheral nervous system, sciatic nerve, lumbar dorsal root ganglion, lumbar dorsal root, lumbar ventral root, cervical dorsal root ganglion, cervical dorsal root, cervical ventral root, sural nerve, tibial nerve, Gasserian ganglion, lumbar spinal cord at swelling, cervical spinal cord at swelling, center of cerebrum, midbrain, cerebellum, pons, medulla oblongota
- Type of staining: uranyl acetate
- Methodology of preparation of sections:
- Thickness: 0.5 micron
- Embedding media: epoxy
- Number of animals evaulated from each sex and treatment group: 6

Statistics:

Body weights and food consumption were analyzed using Bartlett's test, and the Kruskal-Wallis test if needed. Motor activity testing was analyzed using a repeated measures analysis in Snedecor and Cochran (1980). Two-Way Classifications. In Statistical Methods, Iowa State University Press (14) 255-273.

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:

not examined

Clinical biochemistry findings:

not examined

Behaviour (functional findings):

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
One animal died due to a fracture of the muzzle/nares on day 22. No other animals died during the study. Males and females in treatment groups had more staining of the muzzle/head or periorbital region. No other significant differences between control groups and treatment groups were noted.

BODY WEIGHT AND WEIGHT GAIN
Body weight was unaffected by treatment.

FOOD CONSUMPTION
No adverse effects to food consumption were noted.

NEUROBEHAVIOUR
The motor activity results showed no significant differences between control groups and treatments groups. The results of the hindlimb splay and grip strength studies showed no significant differences between control groups and treatment groups.

GROSS PATHOLOGY
No treatment related effects were found.

NEUROPATHOLOGY
No treatment related finding were noted.

Key result

Dose descriptor:

NOAEC

Effect level:

9 000 ppm

Sex:

male/female

Basis for effect level:

other: Neurotoxicity

Key result

Critical effects observed:

no

Conclusions:

Exposure to the test substance had no effect on the behavior of rats. The NOAEC for sub-chronic neurological effects is 9000 ppm in rats.

Executive summary:

This study examined the neurological effects of inhalation exposure to commercial hexane to rats. Rats were exposed to nominal concentrations of 0, 900, 3000, or 9000 ppm for 6 hrs/day, 5 days/week, for 13 weeks. Rats were tested monthly throughout the exposure for hindlimb splay and grip strength. A functional observational battery was also performed regularly. Animals were also examined for clinical signs, body weight, and food consumption.

Results showed no effects to behavior or evidence of toxicity. The NOAEC for sub-chronic neurological effects in rats is 9000 ppm (31680 mg/m³).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

31 680 mg/m³

Study duration:

subchronic

Species:

rat

Effect on neurotoxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

There is no data available for Methylcyclopentane. Data available from a structural analogues, n-Hexane and 2-methylpentane, is used as read across.

In a key study, the neurological effects of inhalation exposure to commercial hexane (53% n-hexane) in rats was examined (API, 1990e; Klimisch score = 2). Rats were exposed to nominal concentrations of 0, 900, 3000, or 9000 ppm for 6 hrs/day, 5 days/week, for 13 weeks. Rats were tested monthly throughout the exposure for hindlimb splay and grip strength. A functional observational battery was also performed regularly. Animals were also examined for clinical signs, body weight, and food consumption. Results showed no effects to behavior or evidence of toxicity. The NOAEC for sub-chronic neurological effects in rats was 9000 ppm (31680 mg/m³).

This study directly informs the DNEL.

Supporting data is also available from a review article published by Jennifer B. Galvin and Gary, Bond in 1999. This article summarizes data available from multiple sub-chronic neurotoxicity studies and the same is presented below.

 

2-Methylpentane and the other hexane isomers do not cause peripheral nervous system damage in experimental animals (Frontali et al.,

1981; Egan et al., 1980; Spencer et al., 1980). Shorter chain alkanes (pentane) and hexane isomers free of n-hexane fail to produce the appropriate metabolite to cause polyneuropathy or peripheral neuropathy (Spencer et al., 1980). Both n-hexane and methyl n-butyl ketone (MBK) are metabolized in animals and humans to the diketone 2,5-hexanedione, which is responsible for the peripheral nervous system damage in both humans and rats. 2-Methylpentane and the other isomers of n-hexane do not follow this metabolic pathway. The urine of the rats treated with 2-methylpentane did not contain 2,5-hexanedione. Experimental metabolism studies have confirmed that the variations in structures among the hexanes produce different metabolites. Frontali et al. (1981) demonstrated that the 24-h urine sample for 2-methylpentane had one metabolite, 2-methyl-2-pentanol.

 

The Threshold Limit Value (TLV) Committee of the American Conference of Governmental Industrial Hygienists (ACGIH, 1991) concurs with Spencer et al. (1980) that the specific diketone metabolites of both n-hexane and methyl n-butyl ketone are responsible for the peripheral nervous system damage and that it is unlikely that isomers of n-hexane such as 2-methylpentane follow this metabolic pathway. The studies in the biomedical literature discussed next confirm this.

 

Frontali et al. (1981) investigated peripheral neurotoxicity and the urinary metabolites in rats of the C5–C7 aliphatic hydrocarbons used as

glue solvents in shoe manufacture in Italy. The concern was the human case reports and epidemiological studies of polyneuropathy occurring in shoe and other leather goods factories and their relationship to the use of C5–C7 aliphatic hydrocarbons. With n-hexane, it had been determined that the ketone metabolites 2,5-hexanedione and 5-hydroxy-2-hexanone were responsible for n-hexane’s polyneuropathy. Light and electron microscopy studies on superficial nerve biopsies showed that exposure to n-hexane caused giant axonal degeneration. Spencer and Schaumburg (1977, 1978) called this “central-peripheral distal axonopathy,” which they demonstrated in rats by inhalation exposures to 500 ppm pure n-hexane. Frontali et al. (1981) designed a study to investigate whether the isomers of hexane also caused this same effect. Therefore, he studied inhalation exposures to n-heptane, 2-methylpentane, 3-methylpentane, n-pentane, cyclohexane, and n-heptane. He varied the exposure regimen for the different chemicals. In the case of 2-methylpentane (98% pure), rats were exposed to 1500 ppm for 9 h/d, 5 d/wk, for 14 wk. There were no signs of neuropathy in any of the animals in the Frontali et al. study on 2-methylpentane. After the exposure period ended, samples of nerves were processed for light microscopy. Sections of teased nerve fibres showed no pathological alterations of tissue from the 2-methylpentane-exposed animals. There was a significant decrease in body weight gain for 2-methylpentane. There were no significant differences in hindlimb spread but there was high individual variability.

 

Rats treated with n-hexane developed the typical giant axonal degeneration. The 24-h urine sample from rats exposed to 1500 ppm

2-methylpentane showed only 1 metabolite, 2-methyl-2-pentanol. There were several urinary metabolites for n-hexane, including the known neurotoxic diketone 2,5-hexanedione. The urine of the rats treated with 2-methylpentane did not contain 2,5-hexanedione.

 

Ono et al. (1981) compared the effect of oral admiration of n-hexane to the oral administration of its hexacarbon isomers on the nerve

conduction velocity in the tails of male rats of the Wistar strain. He used >99% pure n-hexane, 2-methylpentane (isohexane), 3-methylpentane, or methylcyclopentane diluted with olive oil and orally administered in increasing doses over a period of 8 wk. The 2-methylpentane and the other chemicals were administered daily according to the following regimen: 0.4 ml in 0.6 ml olive oil daily for 1–4 wk, 0.6 ml in 0.4 ml olive oil for 4–6 wk, and 1.2 ml in 0.8 ml olive oil for 6–8 wk. Motor nerve conduction velocity, motor distal latency, and mixed nerve conduction velocity were measured in the tail before and after 2, 4, and 8 wk.

 

There were no significant changes of motor nerve conduction velocity in the 2-methylpentane or 3-methylpentane groups and the control in

the Ono et al. (1981) study. There were no significant differences between mixed nerve conduction velocity (distal) in 2-methylpentane (isohexane), 3-methylpentane, or methylcyclopentane groups and the control. In these measures, n-hexane consistently reduced nerve conduction velocity compared with the control. There were no statistically significant differences between the solvent-administered groups and the control in motor distal latency. The distal latency in every group had a tendency to decrease as the rats grew. Only in one parameter, the mixed nerve conduction velocity (proximal), the 2-methylpentane (isohexane) and methylcyclopentane groups were significantly less than the control, and only after 8 wk, with a doubling of the dose during the last 2 wk to approximately 2332 mg/kg/d, which is equivalent to about 8.6 oz daily in a 150-lb human.

 

In the study just described, Ono et al. (1981) concluded that the n-hexane group of rats showed a distinct functional impairment of the

peripheral nerve in the tails of rats. They stated that methylcyclopentane, 2-methylpentane, and 3-methylpentane groups had some significant differences in comparison with the control in the experiment, although these differences were not so distinct as those in the n-hexane group. They then stated that the 3-methylpentane group barely showed any impairment. They reported that the neurotoxicity’s of the three chemicals, if any, were not as severe as n-hexane and ranked them as follows: n-hexane > methylcylopentane >= 2 -methylpentane (isohexane) ~ 3-methylpentane.

 

Ono et al. indicated that 2-methylpentane and 3-methylpentane are reported to be partially metabolized into 2-methylpentanol and

3-methyl-pentanol, respectively, but their further metabolism is not clear. They stated that the neurotoxicity of these chemicals should be clarified by inhalation studies, which was done by Frontali et al. as discussed earlier and published in the same way.

The Ono et al. (1981) study on the effects of oral dosing of the hexane isomers on the peripheral nerves in the tails of rats is of limited value. The dosage regimen was increased substantially over the 8-wk period of the study, doubling between wk 4–6 and wk 6–8, making it difficult to interpret the dose response. The volume of the dosing vehicle, which is known to influence the disposition of a chemical, also was changed over time. Morphological/pathological evaluations were not done to investigate differences between the treated groups and the controls. The results were reported in 1981, and have not been reproduced since that time. The suggestion of possible weak neurotoxicity of 2-methylpentane and 3-methylpentane differs from study results reporting that 2-methylpentane, 3-methylpentane, and hexane isomers free of n-hexane do not produce peripheral nervous system damage in experimental animals (Frontali et al., 1981; Spencer et al., 1980; API, 1982, 1983; Egan et al., 1980).

 

The American Petroleum Institute (API) investigated the neuropathic potential of (1) mixed hexane isomers, (2) mixed hexane isomers

combined with different concentrations of n-hexane, and (3) n-hexane alone. Phase I of this two-part study involved the exposure by inhalation of Sprague-Dawley rats to (1) 125 or 500 ppm n-hexane alone, or (2) 125 ppm n-hexane combined with 125, 375, or 1375 ppm of mixed hexane isomers (API, 1983). In Phase II of the experiment, rats were exposed to 500 ppm of mixed hexane isomers; 500 ppm n-hexane plus 500 ppm mixed hexane isomers; and n-hexane alone (API, 1983). Both Phase I and II exposures were virtually continuous for 22 h/d, 7 d/wk, for up to 6 mo.

 

In Phase I of the API study, the most significant pharmacotoxic sign was the abnormal gait in the group exposed to 500 ppm n-hexane alone.

In Phase II of the study, rats exposed to 500 ppm mixed hexane isomers showed no evidence of nerve and muscle lesions. Rats exposed for approximately 6 mo to 500 ppm n-hexane plus 500 ppm mixed hexane isomers, or to 500 ppm n-hexane alone, developed an abnormal gait, which increased in incidence and severity over time, especially in the 500 ppm n-hexane alone group. These latter two groups had 25 and 30% lower body weight, respectively, than either the controls or the rats exposed to 500 ppm mixed hexane isomers.

 

Microscopic findings on necropsy showed a greater incidence of a trace-to-mild degree of atrophy of the sciatic and/or anterior tibial nerve

with a mild secondary atrophy of skeletal muscle in the 500 ppm n-hexane-treated rats than in the 500 ppm n-hexane plus 500 ppm mixed hexane isomers group. Rats exposed to 500 ppm n-hexane alone or to 500 ppm n-hexane plus 500 ppm mixed hexane isomers showed hindlimb weakness after 2 and 6 mo, respectively. The changes induced by exposure to 500 ppm n-hexane plus 500 ppm mixed hexane isomers were similar in degree to those caused by exposure to n-hexane alone. Neuropathological changes were expressed in both groups exposed at 500 ppm. However, no neuropathological changes were noted at concentrations below 500 ppm (API, 1982).

 

Egan et al. (1980) reported that a mixture of hexane isomers free of n-hexane gave no evidence of neurotoxic effects in rats exposed to 500

ppm for 22 h/d for up to 6 mo. The positive control, methyl n-butyl ketone (MBK), developed peripheral neuropathy after 4 mo of exposure to the same test protocol. The negative control, methyl ethyl ketone (MEK), did not develop polyneuropathy. None of the test animals showed neurological impairment or any other clinical disorders during the exposure period.

 

Source: Jennifer B. Galvin, Gary Bond (1999) 2-METHYLPENTANE (ISOHEXANE), Journal of Toxicology and Environmental Health, Part A, 58:1-2, 81-92, DOI: 10.1080/009841099157449

Justification for classification or non-classification

There is no data available for Methylcyclopentane. Data available from a structural analogues, n-Hexane and 2-methylpentane, is used as read across.

Based on available read across data, Methylcyclopentane does not warrant classification for neurotoxicity under the Regulation (EC) 1272/2008 on classification, labelling and packaging of substances and mixtures (CLP).