Registration Dossier

Administrative data

Description of key information

The NOAEC for subchronic toxicity in mice is 2000 ppm (6,880 mg/m3) (based on haematological changes at 7000 ppm (24,080 mg/m3)).
In both rats and mice the NOEC for acute, transient effects was 500 ppm (1,720 mg/m3).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
90 day
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, published in peer reviewed literature, no restrictions, fully adequate for assessment
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)
Deviations:
not specified
GLP compliance:
yes
Limit test:
no
Species:
mouse
Strain:
other: Crl:CD-1 BR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS- Source: Charles River Laboratories Inc., Raleigh, North Carolina, USA
- Age at study initiation: 39 days
- Housing: Individually in suspended stainless steel, wire-mesh cages with males and females on separate cage racks.
- Diet: PMI Nutrition International, Inc. Certified Rodent LabDiet® 5002 ad libitum (except during exposure)
- Water: ad libitum (except during exposure)
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 23 ± 2°C
- Humidity: 50 ± 10%
- Air changes (per hr): Not reported
- Photoperiod: 12-hour light/dark cycle

IN_LIFE DATES: not reported
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: stainless-steel and glass exposure chambers ( 1.4 m3) with a one-pass, flow-through mode with at least 12 air changes per hour
- System of generation: Atmospheres were generated by heating liquid cyclohexane under nitrogen prior to mixing with filtered, humidified air. The chamber concentration of cyclohexane was controlled by varying the amount of the liquid evaporated in the chamber air stream. Control animals were exposed to air alone.
- Temperature, humidity, pressure in air chamber: During exposure, relative humidity, chamber temperature and air flow rates were monitored and recorded at 15-minute intervals. Chamber oxygen content was measured at least twice daily.

TEST ATMOSPHERE: Analysed by gas chromatography (GC-FID) at approximately 15 minute intervals during each 6 hour exposure period
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were taken from representative areas of the chambers and analyzed with a Hewlett Packard 5880 gas chromatograph equipped with a flame ionization detector. Samples were chromatographed isothermally at 70°C on a HP-20M Carbowax column. The distribution of cyclohexane in the chambers was homogenous. For all 3 studies, the overall mean concentrations of cyclohexane measured in the exposure chambers were 500, 2000, and 7000 ppm (expressed to two significant digits). Additional analyses indicated that the cyclohexane was stable over the duration of the 3 studies and was 99.99% pure.
Duration of treatment / exposure:
13-14 wk. Approximately 90 days (at least 65 days exposure). Subgroups of mice also observed for a 1 month recovery period.
Frequency of treatment:
6 hr/d, 5 d/wk
Remarks:
Doses / Concentrations:
0 (air), 500, 2000, 7000 ppm
Basis:
other: target concentration
Remarks:
Doses / Concentrations:
500, 2000, 7000 ppm
Basis:
other: overall mean measured
No. of animals per sex per dose:
20/sex in the 0 and 7000 ppm groups, 10/sex in the 500 and 2000 ppm groups
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: The exposure concentrations selected for these studies were based on the results of two-week studies in rats and mice, and on the physiochemical properties of cyclohexane (highest concentration reflects 60% of lower explosive limit).
- Post-exposure recovery period in satellite groups: 1 month
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: During each exposure animals visible through the chamber window were checked for obvious signs of distress and for their response to an auditory alerting stimulus. The alerting response was determined prior to the initiation of each exposure, approximately 2, 4, and 6 hours after initiation of exposure, and during the time required to clear the chambers of test substance.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Immediately following removal from the exposure chambers.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: - Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:- Body weight gain in g/food consumption in g from the consumption and body weight gain data: Yes

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Pre-test period and prior to 90 day sacrifice.
- Dose groups that were examined: All groups.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 45 days, 90 days and at end of recovery period
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: 10/sex/dose level
- Parameters examined: erythrocytes, leukocytes, and platelets; haemoglobin concentration, haematocrit, mean corpuscular volume, mean corpuscular Haemoglobin and mean corpuscular haemoglobin concentration; relative numbers of neutrophils, band neutrophils, lymphocytes, atypical lymphocytes, monocytes, eosinophils and basophils.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 45 days, 90 days and at end of recovery period
- Animals fasted: No
- How many animals: 10/sex/dose level
- Parameters examined: total protein

URINALYSIS: No
Sacrifice and pathology:
10 mice/sex/concentration were killed by carbon dioxide asphyxiation and exsanguination, and necropsied. Approximately 1 month later, all surviving mice were similarly killed and necropsied.

Organ weights: The lungs, brain, heart, liver, spleen, kidneys, ovaries, adrenal glands, and testes were weighed and organ weight/body weight and organ weight/ brain weight ratios were calculated.

The following tissues were collected from all animals : skin, bone marrow (sternum and femur), lymph nodes (mandibular and mesenteric), spleen, thymus, aorta, heart, trachea, lungs, nose, larynx, pharynx, salivary glands, oesophagus, stomach, liver, pancreas, small intestine (duodenum, jejunum, and ileum), large intestine (caecum, colon, and rectum), kidneys, urinary bladder, pituitary gland, thyroid - parathyroids, adrenal glands, prostate, mammary glands, testes, ovaries, epididymides, uterus, seminal vesicles, vagina, brain (includes sections of medulla/pons, cerebellar cortex, cerebral cortex), spinal cord (cervical, thoracic, lumbar), sciatic nerve, skeletal muscle, sternum, femur, eyes, exorbital lacrimal glands, Harderian glands, Zymbal's glands, and tibial nerve.

Histopathology: Tissues from animals in the 7000 ppm and control groups and from all animals that were found dead, killed in extremis, or accidentally killed, were examined microscopically. Lungs, liver, kidneys, nose, and relevant gross lesions from animals in the 500 and 2000 ppm groups were also examined microscopically.
Statistics:
Body weight, food consumption, food efficiency, and organ weight data: univariate Analysis of Variance (ANOVA), with Dunnett's Test. Incidence of clinical observations: Cochran-Armitage test for trend and Fisher's exact test. Clinical pathology: ANOVA and Bartlett's test for homogeneity of variances. Dunnett's test was used to compare means from the control groups and test groups. If the results of the Bartlett's test were significant (p < 0.005), nonparametric procedures (Kruskal-Wallis and Mann-Whitney U and Dunn's tests) were used to compare control and treated group means. Separate analyses were performed for each gender and for each dependent variable. Except for Bartlett's test, if the p value associated with the parametric or non-parametric tests was 0.05 or less, the difference was judged to be statistically significant.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY During exposure to 2000 or 7000 ppm, mice had a diminished or an absent response to delivery of a punctate auditory alerting stimulus. Immediately following exposure, 7000 ppm males and females and 2000 ppm females had a compound-related increase in incidence of wet and/or stained fur (mouth, chin, and/or perineum). These signs were transient, were not observed during exposure or prior to exposure the following day, and were not associated with any behavioural or histopathological changes. During exposure, mice exposed to 7000 ppm had clinical signs of toxicity (hyperactivity, circling, jumping/hopping, excessive grooming, kicking of rear legs, standing on front legs, and occasional flipping behaviour). Clinical signs of toxicity observed in 7000 ppm mice immediately after exposure included hyperactivity, hyperreactivity, ruffled fur (females only), gait abnormalities, spasms in both rear legs, and excessive grooming (males only). The clinical signs observed in mice during and immediately after exposure were transient, and were not present prior to the subsequent exposure. A few mice exposed to 2000 ppm appeared hyperactive during exposure in the latter portion of the study.

HAEMATOLOGY Male and female mice exposed to 7000 ppm had slight increases in measures of circulating erythrocyte mass (red blood cells, haemoglobin, haematocrit).

CLINICAL CHEMISTRY Male and female mice exposed to 7000 ppm had slight increases in plasma protein concentration (males only).

ORGAN WEIGHTS Male and female mice exposed to 7000 ppm had significantly increased relative liver weights, and 7000 ppm male mice also had significantly increased absolute liver weights at the end of the exposure period. At the end of the 1-month recovery period, absolute and relative liver weights of male and female mice were similar to control.

Dose descriptor:
NOAEC
Remarks:
(acute transient effects)
Effect level:
500 ppm
Sex:
male/female
Basis for effect level:
other: 1,720 mg/m3 - based on a diminished / absent response to an auditory alerting stimulus during exposure to 2000 ppm and above.
Dose descriptor:
NOAEC
Remarks:
(subchronic toxicity)
Effect level:
2 000 ppm
Sex:
male/female
Basis for effect level:
other: 6,880 mg/m3 - based on haematological changes at 7000 ppm (24,080 mg/m3). This value is very conservative since liver effects at 6,000 ppm upwards could be viewed as adaptive in nature
Critical effects observed:
not specified

Summary of compound-related changes in clinical pathology parameters in male and female mice exposed to cyclohexane

Time point

Dose

Males

Females

Red blood cells

Haematocrit

Haemoglobin

Plasma protein

Red blood cells

Haematocrit

Haemoglobin

Plasma protein

45 day

0 ppm

500

2000

7000

9.29

9.78

9.58

9.96

44

46

45

48

16.0

16.7

16.6

17.9*

6.2

6.2

6.3

6.8*

9.39

9.53

9.26

10.26

45

46

45

50*

16.1

16.6

16.1

18.2$

5.9

6.2

5.9

6.2

90 day

0 ppm

500

2000

7000

9.28

10.14*

10.35*

10.16*

44

48*

48*

49*

16.5

17.5

17.6

17.6

6.1

6.7

6.5

6.5

9.14

9.14

9.11

9.98*

43

44

44

49*

16.1

16.1

15.8

17.4*

5.8

6.1

6.1

6.1

Recovery

0 ppm

500

2000

7000

8.99

ND

ND

9.54

42

ND

ND

45*

15.1

ND

ND

15.9*

6.4

ND

ND

6.5

8.99

ND

ND

9.79*

43

ND

ND

45

15.8

ND

ND

16.6

6.0

ND

ND

6.1

* Statistically significant at p0.05 by Dunnett’s criteria

$ Significantly different from control at p0.05 by Mann-Whitney criteria

Conclusions:
The NOAEC for subchronic toxicity was 2000 ppm (6,880 mg/m3), based on haematological changes at 7000 ppm. The NOAEC for acute, transient CNS effects was 500 ppm (1,720 mg/m3).
Executive summary:

Inhalation studies were conducted to determine the potential toxicity and/or potential neurotoxicity of cyclohexane. Groups of mice were exposed to 0, 500, 2000, or 7000 ppm concentrations of cyclohexane vapour 6 hr/day, 5 days/week for 14 weeks. Subgroups were further observed during a 1-month recovery period. The NOEC for acute, transient effects was 500 ppm (1,720 mg/m3) (based on behavioural changes during exposure at 2000 ppm and above). The NOAEC for subchronic toxicity in mice is 2000 ppm (6,880 mg/m3) (based on haematological changes at 7000 ppm).

 

It is of note that there were no concomitant histological findings associated with the liver weight effects reported at 7,000 ppm. These results were not in accordance with those found during a two-week range finding test (histological findings from 3,000 ppm and above in females and at 9,000 ppm in males). Consequently the reported NOAEC of subchronic toxicity is considered to be very conservative since liver effects at 6,000 ppm upwards could be viewed as adaptive in nature (as also concluded in the RAR, 2004).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
6 880 mg/m³
Study duration:
subchronic
Species:
mouse
Quality of whole database:
Adequate information is available to characterise the repeated inhalation toxicity of cyclohexane in animals.

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Oral

No published data are available.

 

Inhalation

Two relatively recent inhalation studies (with associated range-finding studies) have been conducted to determine the potential toxicity and/or potential neurotoxicity of cyclohexane. The 90-day studies reported by Malley et al (2000) are selected as the key studies on the basis of reliability and on the conclusions from regulatory review (SCOEL, 2001; RAR, 2004). In both studies, groups of rats and mice were exposed to 0, 500, 2000, or 7000 ppm concentrations of cyclohexane vapour 6 hr/day, 5 days/week for 14 weeks. Additional groups were retained and observed for a 1-month recovery period. Doses were based on preliminary studies consisting of 9 exposures over 2 weeks at concentrations of 3000, 6000 or 9000 ppm.

Slight liver effects were induced after 14 or 90 day exposure periods. Increases in absolute and relative liver weight and centrolobular hypertrophy were noted in both rats and mice at dose levels between 6000 and 7000 ppm (in the preliminary and the 90-day studies, respectively). The NOAEC for hepatic effect is estimated to be 2000 ppm (6,880 mg/m3). It was concluded in the RAR (2004) that this value is very conservative since liver effects at 6,000 ppm upwards could be viewed as adaptive in nature.

 

For neurotoxicity (also see CSR Section 5.10), the same studies assessed narcotic properties and motor activity changes. In mice and rats, reversible changes of responses to stimulus (decreases or no reactions) were observed at doses of 6,000 ppm in the preliminary studies and at doses of 2,000 ppm in the 90-day studies. It is concluded that this effect is an acute effect and the NOAEC is 500 ppm (1,720 mg/m3). This value will be taken into account in the risk characterisation of acute neurotoxicity.

 

In an older study in rabbits (Treon et al, 1943), liver and renal effects were observed at dose levels of about 800 ppm, leading to a NOAEL of about 500 ppm. However previous review of this study (RAR, 2004) concluded that, due to limitations of the study, these effects should not be taken into account in the risk characterisation.

 

Dermal

The RAR (2004) reviewed a repeat dose rabbit study (Treon et al, 1943) where a single rabbit received repeated daily applications of undiluted cyclohexane on uncovered skin over a period of 14 days, giving a total dose of 180.2 g/kg. The result was irritation and thickening of the skin but no fatality. It was not possible to determine a dermal NOAEL from this limited study for reliability reasons.

 

The applicant submits that this endpoint is waived in accordance with column 2 of REACH Annex IX as inhalation is the main route of exposure, this study is not considered relevant for consideration in hazard or risk assessment.

 

Other routes

The RAR (2004) reviewed a study by Bernard et al. (1989) which evaluated the nephrotoxicity of cyclohexane in female rats in which the authors suggested that cyclohexane nephrotoxicity was due to cyclohexanol. The applicant submits that this study is considered unreliable for hazard identification or characterization because it utilized a non-relevant route of exposure (intraperitoneal injection), and therefore it is not considered further in this context.

 

Human data

Human exposure data is reviewed in the RAR (2004). It has been reported that cyclohexane exposure at the concentrations up to 274 ppm (959 mg/m3)) did not induce induced any significant increase in the prevalence of subjective symptoms or measured biochemical parameters of liver and kidney function (Yasugi et al., 1994).

A 1991 UK HSE review pointed out that cyclohexane, associated with other chemicals, has been implicated by some authors in the peripheral neuropathy reported among in workers in Italian shoe factories, printing plants and in paint workers; cyclohexane frequently being a major component of the solvents and adhesives used (De Rosa et al., 1985; Mutti et al., 1982; Franco et al., 1979). However, such workers received mixed exposure to various solvents, including n-hexane, and HSE concluded from the experimental evidence that the neuropathy was most likely caused by the n-hexane component of the solvents and adhesives used in these industries (as cited in the RAR, 2004).

The neurotoxicity of cyclohexane during occupational exposure in a luggage factory of 18 women (aged from 18 to 56) who were exposed for eight hours a day to glue containing 75.6% cyclohexane, 12% toluene and 0.9% n-hexane was assessed in a study was carried out by Yuasa et al. (1996). Overall, there was no evidence of neurotoxicity during a relatively short period of exposure at low concentrations (below the time weighted average threshold limit values of the US (150 ppm)).

 



Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
The NOAEC for subchronic toxicity in mice is 2000 ppm (6,880 mg/m3), based on haematological changes at 7000 ppm (24,080 mg/m3)

Repeated dose toxicity: inhalation - systemic effects (target organ) cardiovascular / hematological: other

Justification for classification or non-classification

These effects are considered to warrant labelling under CLP: Specific Target Organ Toxicity - Single exposure, Category 3 following the criteria for narcotic effects and assign with H336.