Reaction mass of 4-vinylcyclohexene, cyclohexane and n-hexane

Repeated dose toxicity: inhalation

Administrative data

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

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2000

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Cyclohexane

EC Number:

203-806-2

EC Name:

Cyclohexane

Cas Number:

110-82-7

Molecular formula:

C6H12

IUPAC Name:

cyclohexane

Details on test material:

- Name of test material (as cited in study report): cyclohexane
- Physical state: clear colourless liquid with a mild, sweet odour
- Analytical purity: >99.9%

Test animals

Species:

rat

Strain:

other: Crl:CD 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: 27 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)
- Acclimatisation 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

Administration / exposure

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 also observed for a 1 month recovery period.

Frequency of treatment:

6 hr/d, 5 d/wk

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0 (air), 500, 2000, 7000 ppm
Basis:
nominal conc.

Doses / concentrations 2

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 a two-week preliminary study and on the physiochemical properties of cyclohexane (highest concentration reflects 60% of lower explosive limit).
- Post-exposure recovery period in satellite groups: 1 month

Examinations

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: Yes (approximately 14 hours prior to collection)
- 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: Yes (approximately 14 hours prior to collection)
- How many animals: 10/sex/dose level
- Parameters examined: alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, sorbitol dehydrogenase, gamma-glutamyl transpeptidase, creatine phosphokinase, lactate dehydrogenase; concentrations of glucose, urea nitrogen, calcium, phosphate, bilirubin, cholesterol, creatinine, triglycerides, total protein, albumin, globulin, sodium, potassium and chloride.

URINALYSIS: Yes
- Time schedule for collection of urine: No data
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters examined: volume, osmolality, urobilinogen, pH, haemoglobin, glucose, protein, bilirubin, and ketone

Sacrifice and pathology:

10/sex/concentration killed by carbon dioxide asphyxiation and exsanguination, and necropsied. Approximately 1 month later, all surviving rats 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, 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 used to compare control and test group means. If Bartlett's test significant (p < 0.005), nonparametric procedures (Kruskal-Wallis and Mann-Whitney U and Dunn's tests) used. Separate analyses were performed for each gender and for each dependent variable. Except for Bartlett's test, if p≥ 0.05, the difference was judged to be statistically significant.

Results and discussion

Results of examinations

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:

no effects observed

Clinical biochemistry findings:

no effects observed

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:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY During exposure to 2000 or 7000 ppm, rats had a diminished response or an absent response to delivery of a punctate auditory alerting stimulus. Immediately following exposure, 7000 ppm males and females and 2000 ppm females displayed a compound-related increase in the 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 morphological changes.

ORGAN WEIGHTS Male rats exposed to 7000 ppm had significantly increased relative liver weights at the end of the exposure period. At the end of the 1-month recovery period, relative liver weights of 7000 ppm male rats continued to be significantly higher.

HISTOPATHOLOGY Male and female rats exposed to 7000 ppm had a significantly increased incidence of hepatic centrilobular hypertrophy at the end of the exposure period, which was not observed at the conclusion of the 1-month recovery period.

Effect levels

1

Dose descriptor:

NOAEC

Remarks:

rat (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.

2

Dose descriptor:

NOAEC

Remarks:

(subchronic toxicity)

Effect level:

7 000 ppm

Sex:

male/female

Basis for effect level:

other: 24,080 mg/m3 - based on the lack of adverse effects on body weight, haematology, clinical chemistry and tissue histopathology.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The NOAEC for subchronic toxicity was 7000 ppm (24,080 mg/m3), based on an absence of adverse effects on body weight, haematology, clinical chemistry, tissue histopathology. The increased liver weights and centrilobular hepatocellular hypertrophy observed in males only at 7000 ppm is an adaptive physiological change considered not to be an adverse systemic effect. The NOAEC for acute, transient CNS effects was 500 ppm.

Executive summary:

Inhalation studies were conducted to determine the potential toxicity and/or potential neurotoxicity of cyclohexane. Groups of rats 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 no-observed-adverse-effect concentration (NOAEC) for acute, transient effects was 500 ppm (1,720 mg/m³) (based on a diminished/absent response to an auditory alerting stimulus at 2000 ppm (6,880 mg/m³) and above). The NOAEC for subchronic toxicity in rats was 7000 ppm (24,080 mg/m³) (based on the lack of adverse effects on body weight, haematology, clinical chemistry and tissue histopathology).