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Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
not specified
Remarks:
no historical control data; whole body exposure without justification
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Cyclohexanone
EC Number:
203-631-1
EC Name:
Cyclohexanone
Cas Number:
108-94-1
Molecular formula:
C6H10O
IUPAC Name:
cyclohexanone
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source: ACROS, Netherlands
- Lot no.: SNGYB and B2XGG
- Purity: 99.7% (SNGYB) and 99.6% (B2XGG)

Test animals

Species:
other: mouse and rat
Strain:
other: B6C3F1 and F344
Details on species / strain selection:
Species were selected for the study because these animals are generally used in subchronic toxicity studies and the availability of considerable
background information for these species.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Japan SLC, Inc. (Shizuoka, Japan)
- Age at study initiation: 6 weeks
- Housing: single
- Diet: provided rodent diet
- Water: filtered tap water
- Acclimation period: 1 week

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 50 +/- 20
- Air changes (per hr): 10-20
- Photoperiod (hrs dark / hrs light): not specified

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
clean air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: LVG-04-A, HCT Co., Seoul, Korea
- Method of concentration analysis: valve control system (VCS-06, HCTm, Seoul, Korea) and GC (Trace 1300, Thermo Fischer Scientific Co., Waltham, MA)
- System of generating particulates/aerosols: saturated vapor was evaporated by bubbling clean air and using a hot water bath.

TEST ATMOSPHERE
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Cyclohexanone concentrations in the chambers were automatically recorded through the valve control system (VCS-06, HCTm, Seoul, Korea) by using a pump and analyzed by gas chromatography (Trace 1300, Thermo Fisher Scientific Co., Waltham, MA) at 30-min intervals during the exposure period.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 h/day, 5 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
100 ppm
Remarks:
equivalent to ca. 408 mg/m³
Dose / conc.:
250 ppm
Remarks:
equivalent to ca. 1020 mg/m³
Dose / conc.:
625 ppm
Remarks:
equivalent to ca. 2550 mg/m³
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The highest exposure concentration of 650 ppm was selected based on the results of an acute inhalation toxicity study (Smyth et al., 1969;mOECD, 1996) which reported 1/6 rats exposed to 2000 ppm for 4 h died and a 4-week repeated inhalation toxicity study (Lee et al., 2018) which reported no effects at exposure concentrations up to 250 ppm in B6C6F1 mice. In addition, conditions of generation of cyclohexanone before starting the study were checked, and it was confirmed that the highest concentration that can be stably and continuously generated at the facility was 650 ppm.
Positive control:
no positive control included

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes / No / Not specified
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: weekly

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: start of treatment and in the last week of exposure period
- Dose groups that were examined: all animals (start) and control and high dose (last week)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: No
- How many animals: All surviving animals
- Checked parameters: leucocyte, platelet (PLT) count,
erythrocyte count, hemoglobin, hematocrit, mean corpuscular
volume, mean corpuscular hemoglobin, mean corpuscular
hemoglobin (MCH) concentration, prothrombin time
(PT, mice only), and activated partial thromboplastin time
(mice only).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At necropsy
- Parameters checked: glucose, total
bilirubin, blood urea nitrogen (BUN), potassium, total protein,
calcium, albumin (ALB), chloride, creatinine, inorganic
phosphorus, total cholesterol (TCHO), sodium, triglyceride,
aspartate aminotransferase (AST), alanine aminotransferase
(ALT), alkaline phosphatase (ALP), and c-glutamyl transpeptidase

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes.
- Organs that were weighed: adrenal glands, ovaries (female only), brain, spleen, epididymides (male only), testes (male only), heart, thymus, kidneys, uterus (female only), liver, and lung

HISTOPATHOLOGY: Yes
- Organs checked: ovaries (female only), adrenal glands, pancreas, parathyroid glands, aorta (thoracic), pituitary gland, brain, prostate (male only), bone marrow, rectum, cecum, salivary glands, colon, sciatic nerve, duodenum, seminal vesicle (male only), epididymides (male only), skeletal muscle, esophagus, skin, eyes (with optic nerve), spinal cord (cervical, thoracic, lumbar), femur (F-T joint), spleen, gall bladder (mice only), Harderian glands, sternum, heart, stomach, ileum, teeth, jejunum, testes (male only), kidneys, thymus, larynx, thyroids, liver, trachea, lung, urinary bladder, lymph node, uterus with cervix (female only), mammary gland (female only), vagina (female only), and nasopharyngeal tissue.
- In addition to histopathological examination of the high exposure group of animals,
histopathological examination of the skin (mice only), liver (rats only) and kidney (rats only) was performed for all animals of the low- and medium-concentration groups.
Statistics:
Homogeneity of the variances: Levene test
When variances were homogeneous: one-way ANOVA
Differences between control and treated groups: Dunnett's test
Heterogeneity of the variances: Kruskal-Wallis test
Differences between control and treated groups: Dunn's rank sum test

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
RATS
no effects observed

MICE
In all test groups, loss of hair was noted
Mortality:
mortality observed, non-treatment-related
Description (incidence):
RATS
no mortality observed

MICE
625 ppm: two males died
control: one female died
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
RATS
625 ppm: MCH in males decreased, PLT count increased, PT decreased in females

MICE
625 ppm: increased reticulocyte counts in males
250 ppm: increased leukocyte (WBC) counts in males
100 ppm: PLT count decreased in males
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
RATS
625 ppm: AST and ALT increased in males, and increased ALT in females. BUN levels increased in males. ALP levels decreased in females
250 ppm: AST and ALT increased in males, ALP levels increased in males

MICE (effects observed, non-treatment-related)
625 ppm: decreased total bilirubin levels in males, BUN levels increased in males, TCHO increased in females
250 ppm: decreased ALP levels in males
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
RATS
625 ppm: absolute and relative weights of the liver in males increased, relative weight of spleen in males increased, absolute weight of kidney increased in males
250 ppm: relative weight of the liver in males increased, relative weight of spleen in females increased
Females did not show a statistical significance increase in liver weights but showed a dose-dependent increase

MICE (effects observed, non-treatment-related)
650 ppm: absolute and relative liver weights increased in males, absolute liver weight increased in females, absolute kidney weight increased in males
250 ppm: relative kidney weight increased in females
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
RATS
625 ppm: white focal lesions in lungs of males
250 ppm: adhesions in the accessory lobe of the liver and right kidney in females

MICE
625 ppm: In two dead males, a black spot lesion of the stomach, small of spleen, and thymus were observed. A nodule was noted in thoracic muscle of one male
Control: small of the thymus in a dead female

No abnormal findings related to the test chemical were observed in the animals that survived until the scheduled terminal necropsy.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
RATS
625 ppm: indicative of alveolar macrophage aggregation in males, four cases of bile duct hyperplasia in the liver of male animals
250 ppm: adhesion of hepatocytes to the renal capsule and atrophy of the glomeruli and tubules in the renal cortex in females. Two cases of bile duct hyperplasia in the liver of male animals

Males showed hyaline droplet accumulation in the distal convoluted tubule and cortical tubular basophilia in the kidney. Hyaline droplet accumulation was observed in all test groups including the control groups. However, the extent increased in a concentrationdependent manner.
Tubular basophilia in the renal cortex had known a spontaneous lesion due to aging in the rats and was exacerbated by the test substance. The severity of tubular basophilia increased upon exposure to cyclohexanone in a dosedependent manner. Therefore, it was considered that tubular basophilia in the renal cortex may be aggravated by the progression of chronic progressive nephropathy due to cyclohexanone exposure

MICE (effects observed, non-treatment-related)
625 ppm: atrophy of the liver, spleen, and thymus and single-cell necrosis of the liver and erosion of the glandular stomach in the dead males
Control: atrophy of the liver, spleen, and thymus and single-cell necrosis of the liver in the dead female

No abnormal findings related to the test chemical were observed in the animals that survived until the scheduled terminal necropsy.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Remarks:
rats
Effect level:
100 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical biochemistry
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Remarks:
mice
Effect level:
> 625 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed

Target system / organ toxicity

open allclose all
Critical effects observed:
yes
Lowest effective dose / conc.:
250 ppm
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Critical effects observed:
yes
Lowest effective dose / conc.:
250 ppm
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes

Any other information on results incl. tables

Mean ± standard deviation values for the cyclohexanone concentrations in the inhalation chambers were 99.51 ± 4.63 ppm, 252.87 ± 8.38 ppm, and 625.32 ± 35.48 ppm for rats, and 103.63 ± 26.97 ppm, 251.36 ± 38.49 ppm, and

617.83 ± 86.41 ppm for mice throughout the 13-week exposure period. Deviations in the mean observed concentrations from the target concentrations were less than 11.5%.

Applicant's summary and conclusion

Conclusions:
In conclusion, inhalation exposure to cyclohexanone vapors for 13 weeks induced hepatoxicity and renal toxicity in rats of both sexes. The authors concluded on a NOAEL of 100 ppm for the hepatic endpoint in F344 rats and >625 ppm in B6C3F1 mice.
Executive summary:

In this study, ten male and ten female rats (F344) and mice (B6C3F1) per group were exposed to cyclohexanone vapors at 100, 250, and 625 ppm concentrations for 6 h per day, 5 d per week, and for 13 weeks. Clean air was used as negative control. All rats and mice were killed after the exposure period. Clinical signs, body weight, feed intake, and ophthalmoscopy findings were recorded during the exposure period, and hematology, blood biochemistry, organ weights, gross findings, and histopathology were evaluated thereafter.

The following effects were recorded in cyclohexanone-exposed F344 rats: increased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, increased liver weight, and bile duct hyperplasia in the males exposed to 250 and 625ppm cyclohexanone, increased ALT levels and bile duct hyperplasia in the females exposed to 625ppm cyclohexanone, and increased blood urea nitrogen (BUN) and tubular basophilia in the renal cortex in the males exposed to 625 ppm cyclohexanone.

B6C3F1 mice exposed to cyclohexanone showed no obvious exposure-related effects.

Based on the findings in male rats, the NOAEL (rats) was set at 100 ppm and the NOAEL (mice) greater than 625 ppm.

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