N-hexane

Neurotoxicity

Description of key information

LOAEC (sub-chronic, rat; conduction velocity) = (10560 mg/m³) 3000 ppm 

Occupational epidemiology data describing neurotoxicity effects in humans are avaible for n-hexane and have been extensively reviewed by WHO, ATSDR, and U. S. EPA.

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:

1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: This study is classified as reliable with restrictions because although a GLP statement was provided limited data on methods and guidelines were reported. The study seemed to be well-conducted.

Qualifier:

no guideline followed

Principles of method if other than guideline:

7 rats were exposed to 3000 ppm of hexane vapors for 12 hrs a day for 16 weeks. Body weights and conduction velocity of the peripheral nerve of the tail was measured at 0, 4, 8, 12 and 16 weeks. At the end of the exposure period, two animals were sacrificed and the nerve tissue examined.

GLP compliance:

yes

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 308 +/- 18 g

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23.5-24.5

Route of administration:

inhalation: vapour

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Temperature, humidity, pressure in air chamber: 23.5-24.5 degree C, 41-61% humidity

TEST ATMOSPHERE
- Brief description of analytical method used: gas detector measurements were taken daily, liquid chromatography measurements were taken twice weekly

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

gas detector measurements were taken daily, liquid chromatography measurements were taken twice weekly

Duration of treatment / exposure:

16 weeks

Frequency of treatment:

daily, 12 hrs per day

Remarks:

Doses / Concentrations:
3000 ppm
Basis:
no data

No. of animals per sex per dose:

7

Control animals:

yes, sham-exposed

Observations and clinical examinations performed and frequency:

BODY WEIGHT: Yes
- Time schedule for examinations: 0, 4, 8, 12, and 16 weeks after start of exposure

OTHER: The conduction velocity of the peripheral nerve of the tail was measured at 0, 4, 8, 12 and 16 weeks

Sacrifice and (histo)pathology:

HISTOPATHOLOGY: Yes, 2 rats were sacrificed at the end of 16 weeks, and their gastrocnemius and soleus muscles, dorsal trunk of the tail nerve, and the tibial nerve examined.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

not examined

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):

effects observed, treatment-related

Gross pathological findings:

not examined

Neuropathological findings:

effects observed, treatment-related

Other effects:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY
Two animals died during the study. One animal died 1 day before the end of the exposure period, and one animal died three days before the end of the exposure period.

BODY WEIGHT AND WEIGHT GAIN
Body weight gain was significantly reduced at 4 weeks after start of exposure, and remained depressed for the rest of the experiment.

NEUROBEHAVIOUR
Unsteady gait was observed in one animal at 10 weeks exposure, and in 4 animals at 12 weeks. 2 animals at this time point also showed foot drop. At 16 weeks exposure, the five surviving rats had unsteady gait, and two had foot drop. All animals had muscular atrophy at this time point.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were paranodal swellings in the myelinated fibers of the tibial nerve and dorsal trunk of the tail nerve. There were an excessive number of neurofilaments, vesicles, multivesicular bodies, mitochondria, myelin figures, and dense bodies in the paranodal axoplasm and no neurotubules. Denervated neuromuscular junctions in the muscles were observed. Muscle fibers were of irregular shape and size, and had an increased number of nuclei, and had disordered myofilaments, zig-zagging of the z-band, and invaginations of the plasma membrane.

OTHER FINDINGS
The motor nerve conduction velocity (MCV) was significantly less than controls by 4 weeks of exposure. MCVs could not be measured in 2 animals after 16 weeks due to nerve damage. Distal latencies (DL) were significantly prolonged after 4 weeks of exposure, and could not be measured in 2 aminals at 16 weeks of exposure.

Key result

Dose descriptor:

LOAEC

Effect level:

3 000 ppm

Sex:

male

Basis for effect level:

other: reduced body weight gain

Remarks on result:

other:

Conclusions:

The LOAEC for sub-chronic exposure to hexane vapors was 3000 ppm (10560 mg/m^3) based on reduced body weight gain, mortality and neurological effects.

Executive summary:

In this study, 7 rats were exposed to 3000 ppm of hexane vapors for 12 hrs a day for 16 weeks. Body weights and conduction velocity of the peripheral nerve of the tail was measured at 0, 4, 8, 12 and 16 weeks. At the end of the exposure period, two animals were sacrificed and the nerve tissue examined. Two animals died before the end of the exposure period. All animals showed reduced weight gain after 4 weeks of exposure. Neurological effects were seen beginning at 10 weeks exposure. Motor nerve conduction velocity and distal latency were significantly affected after 4 weeks exposure. Examination of neural tissue showed damage to the tibial nerve and dorsal trunk of the tail nerve. The LOAEC for sub-chronic exposure was 3000 ppm (10560 mg/m^3). No NOAEC was found.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEC

10 560 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

1 key substance specific study available for assessment

Effect on neurotoxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A sub-chronic neurotoxicity study in rats is available for n-hexane. Additionally, occupational epidemiology data describing neurotoxicity effects in humans are available for n-hexane and have been extensively reviewed by WHO, the Agency for Toxic Substances and Disease Registry (ATSDR), and the U.S. Environmental Protection Agency (U.S. EPA).

n-Hexane, neurological effects in animals

In a key neurotoxicity study, seven rats were exposed to 3000 ppm of n-hexane vapors for 12 hrs a day for 16 weeks (Takeuchi, 1980; Klimisch score =2). Body weights and conduction velocity of the peripheral nerve of the tail was measured at 0, 4, 8, 12 and 16 weeks. At the end of the exposure period, two animals were sacrificed and the nerve tissue examined. Two animals died before the end of the exposure period. All animals showed reduced weight gain after 4 weeks of exposure. Neurological effects were seen beginning at 10 weeks exposure. Motor nerve conduction velocity and distal latency were significantly affected after 4 weeks exposure. Examination of neural tissue showed damage to the tibial nerve and dorsal trunk of the tail nerve. The LOAEC for sub-chronic exposure was 3000 ppm (10560 mg/m³). No NOAEC was found.

n-Hexane, neurological effects in humans

Among hydrocarbon solvents, a unique toxicological effect of n-hexane in humans is its association with pathological changes in the peripheral nervous system, more specifically an axonal neuropathy of the lower extremities.  Development of the axonal neuropathy is the consequence of exposure to a neurotoxic metabolite of n-hexane; 2,4-hexanedione.  Although questions have been raised regarding the potential for other hexane solvents, including other C6 isomers, to produce similar effects, it has been shown, primarily through animal models, that hydrocarbons other than n-hexane do not form the 2,5-hexanedione metabolite and, accordingly, do not produce similar neurological effects (see for example WHO, 1991; ATSDR, 1999, U.S. EPA, 2005).

The association of n-hexane exposure with the development of peripheral neuropathy was first described in studies of Japanese sandal workers.  Based on assessment reviews of epidemiology studies of n-hexane (e.g., WHO, 1991, ATSDR, 1999, U.S. EPA, 2005) exposure estimates ranged from 1625 -8125 mg/m3 (500 – 2500 ppm), although these measurements were typically done after the fact.  As additional complications, these workers typically worked very long hours and often lived in the same location.  The cases were divided into 3 categories based on severity of the disease:

            Group 1        Sensory neuropathy

            Group II        Sensorimotor neuropathy

            Group III       Motor neuropathy with amyotrophy

Studies of 95 employees at an Italian shoe factory population (Mutti et al., 1982) identified subclinical neuropathy, particularly significant reductions in nerve conduction velocity.  The approximate average exposure to n-hexane in the work place was 317 mg/m3 (90 ppm) over a 2 year period.  Additionally, other solvents were used.  When these workers were subdivided into “low” (approximately 243 mg/m3) and “high” (approximately 475 mg/m3) exposure groups based on n-hexane levels, there appeared to be a dose-related trend. 

A study of Taiwanese press proofers was reported by Wang et al. (1986).  Fifteen cases of overt peripheral neuropathy were diagnosed among 59 workers distributed among 16 factories.  The average levels of n-hexane exposure at the two factories, accounting for 8 of the 15 cases, were 669 mg/m³ and 74 mg/m³ respectively.  It was also reported that workers typically worked more than 8 hours/day, and 12 of the 15 cases slept in the factories overnight.  Exposure to n-hexane air concentrations less than 352 mg/m³ (100 ppm) was not associated with clinical peripheral neuropathy.

Due in part to concerns about the quality of the exposure measurements and differences in exposure periods, it has been difficult to establish thresholds for human effects.  One recommendation is to use data on urinary levels of 2,5-hexanedione as a biomarker of exposure (SCOEL, 1995).  Governa et al. (1987) reported that subclinical neurological effects were associated with urinary 2,5 hexanedione concentrations of > 7.5 mg/L whereas there were few effects among individuals with urinary levels 7.5 mg/L.  Based data from Perbellini et al. (1981) relating urinary excretion of n-hexane metabolites with external n-hexane exposure, 7.5 mg/L corresponds to approximately 250 mg/m³ (70 ppm).

Assessment Review References:

ATSDR (Agency for Toxic Substances and Disease Registry) (1999).  Toxicological Profile for n-Hexane.  ATSDR, Atlanta, Georgia.

SCOEL (Scientific Committee on Occupational Exposure Limits) (1995). Recommendations from Scientific Expert Group on Occupational Exposure Limits for n-hexane. SEG/SUM/52C.

U.S. EPA (U.S. Environmental Protection Agency) (2005). Toxicological Review of n-Hexane (CAS No. 110 -54-3) In Support of Summary Information on the Integrated Risk Information System (IRIS). November 2005, U.S. Environmental Protection Agency Washington, DC. EPA/635/R-03/012.

WHO, International Program on Chemical Safety (1991). Environmental Health Criteria (EHC) 122, n-Hexane. World Health Organization, Geneva, Switzerland.

Justification for classification or non-classification

Based on the information presented in the study on anaesthetic activity of n-hexane, n-hexanes are classified as STOT Single Exp. 3 (H336: May cause drowsiness or dizziness) in accordance with CLP EU Regulation 1272/2008.