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Carcinogenicity

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Description of key information

In a chronic drinking water study with rats conducted by Lijinski and Kovatch (1986), groups of male and female F344 rats were treated with cyclohexanone in acidified drinking water at concentrations of 0, 3300, or 6500 ppm for 2 years. The survival of the rats in the high dose groups was slightly reduced, body weight gains decreased with increasing dose. At concentrations of 3300 ppm, 13 % of the male rats developed adenomas of the adrenal cortex. This type of tumors was not found in the females and in only 1/51 male animal of the high dose group. In the male rats of the high dose group there was an increase in follicular tumors of the thyroid.


The reason for the increased incidence of adrenocortical adenomas in male rats of the low dose group has not been discussed by the authors. However, it seems unlikely that the tumor development is treatment-related, as there was no increased incidence of this type of tumors among high-dose male animals. No signs of organotoxic or cytotoxic effects in either the low dose group or the high dose group have been described by the authors.


The other effect described in rats was a marginal increase in the incidence of thyroid tumors among high-dose male rats. This might be a treatment-related effect, although with low statistical significance and not observed in female rats or in male/female mice. This effect has not been commented by the authors of the study.


As described in chapter 7.1 (Toxicokinetics) of the IUCLID file for cyclohexanone, the metabolism of cyclohexanone is different in rats and humans. In the rat, cyclohexanone is reduced to cyclohexanol with subsequent glucuronidation and excretion. In contrast to the metabolism in rats (and dogs), in humans, the glucuronidation pathway is less important; the major part of cyclohexanone gets ring-hydroxylated to 1.2- and 1.4 -diols which are excreted in glucuronidated and non-glucuronidated form.


It is well known that thyroid hormones T3 and T4 are excreted biliary after glucuronidation. It seems likely, that chronic exposure to cyclohexanone leads to an increase in the activity of UDP-glucuronosyltransferases (UGTs), due to an increase in metabolizing capacity based on the chronic exposure to cyclohexanone. This increase in UGT-activity then may lead to an increase in the excretion of T3 and T4, causing a stimulation of thyroid tissue by TSH (thyroid-stimulating hormone) through the pituitary-thyroid axis [1]. As the glucuronidation only takes place partly during the metabolism of cyclohexanone in humans, consequently, T3 and T4 may be excreted to a lesser extent than in rodents. In addition to that, the biological half-lives of T3 and T4 are substantially longer in human plasma than in rat plasma, due to a high-affinity binding to plasma proteins in humans, which is not present in rodents [2]. Therefore, an increase in UGTs in humans in not as critical as in rodents. Basal serum levels of TSH are higher in rats than in humans, and higher in male rats than in female animals [2], which can be an explanation why tumors of the thyroid were only found in male animals. In addition to that, the male rat is more susceptible than female rats or humans to develop hyperplastic and/or neoplastic nodules in response to chronic TSH stimulation [3].


Taking together all the information above, it is questionable that the higher incidence of thyroid tumors in the high-dose male rat is of relevance for humans.


 References: 


[1] Klaassen, C.D., Hood, A.M 2001: Effects of microsomal enzyme inducers on thyroid follicular cell proliferation and thyroid hormone metabolism.Toxicologic Pathology,29, No. 1, 34-40 (2001)


[2] Doehler, K.D., Wong, c.c., von zur Mühlen, a. 1979: The rat as a model for the study of drug effects on thyroid function: consideration of methodological problems.Pharmacological Therapy,5, 305-318 (1979)


[3] Capen, C.C. 1997: Mechanistic data and risk assessment of selected toxic end points of the thyroid gland.Toxicologic Pathology,25, No. 1, 39-48 (1997)


 


In the mouse study, conducted by the same authors (1986), groups of B6C3F1 mice were treated with cyclohexanone in acidified drinking water at concentrations of 0, 6500, or 13000 ppm for males and 0, 6500, 13000, or 25000 ppm for female mice for 2 years. Evaluated endpoints were the same as reported for the rat study. Survival among male mice given 13000 ppm cyclohexanone was reported as 80% at week 90 and about 70% at the end of the study. In female mice, however, only less than 15% animals of the high dose group survived until the termination of the study. After 90 weeks, about 40% females of the mid dose were still alive and survived until study termination. Survival among the low dose rats of both sexes was comparable to the survival rates of control animals. The average bodyweight of male and female mice of the highest dose groups was about 15-20% lower than those of control animals during most of the study. Bodyweights of female mid-dose mice were only marginally depressed and among low-dose female and male animals comparable to those of controls.


Lymphoid hyperplasia, lymphocytic inflammatory infiltrates, and/or malignant lymphoma were observed among most cyclohexanone-treated as well as in control female mice. These effects might be a confounding factor when it comes to interpretation of the other effects observed in female mice during this study. In addition to that, effects can hardly be interpreted due to the poor survival rates of female mice. Lymphomas are a common finding in female B6C3F1 mice, with increasing rates in older animals. 


The incidence of combined benign and malignant hepatocellular neoplasms was significantly higher among low-dose females compared to controls. However, no significant increases in tumor incidence were observed among high-dose males or among high-dose and mid-dose females, which makes a biological relevance of those findings questionable. Moreover, there is a high spontaneous background incidence of liver tumors in mice in chronic studies.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Referenceopen allclose all

Endpoint:
carcinogenicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
GLP compliance:
not specified
Specific details on test material used for the study:
Analysis of the sample showed 96% purity, together with 3% water and 1% unidentified compounds.
Species:
mouse
Strain:
other: C57BL/6xC3H)F1
Sex:
male/female
Details on test animals or test system and environmental conditions:
B6C3F1 mice of both sexes were 7-8 weeks old at the beginning of treatment.
The weight of the male mice averaged 23 g (20-26 g); that of the female mice, 20 g (17-23 g). In the chronic study, mice were housed 4 to a cage.
The study with both species (rats and mice; see repeated dose toxicity) was conducted in a single animal holding room at 22-24°C in which there was frequent change of air (15 times per h). The animals were raised and housed behind a barrier, in a facility with clean-unclean corridors, the air pressure within the room being arranged so that air from the room would flow lo the unclean side. Mice were monitored for murine virus antibodies. The animals were fed Wayne Sterilizable Lab Meal ad libitum.
Controls received only acidified water and were kept in the same room. Each animal was weighed once a week and at the end of the study. Animals were sacrificed and dissected, the organs and tissues were fixed in Formalin, and slides were prepared for histopathological examination.
Route of administration:
oral: drinking water
Vehicle:
water
Remarks:
water was acidified with HCl to prevent bacterial growth.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of the solution at intervals and after 28 days showed that there was no detectable decomposition during this time.
Duration of treatment / exposure:
104 weeks
Frequency of treatment:
continuously
Post exposure period:
none
Dose / conc.:
6 500 ppm
Remarks:
male and female animals (nominal in water)
Dose / conc.:
13 000 ppm
Remarks:
male and female animals (nominal in water)
Dose / conc.:
25 000 ppm
Remarks:
female animals (nominal in water)
No. of animals per sex per dose:
Each treatment group consisted of 50 or 52 male and 50 or 52 female mice, except 47 male mice treated with the highest dose and 41 female mice treated with the highest dose.
Control animals:
yes, concurrent no treatment
Details on study design:
Male mice were given either the MTD concentration of cyclohexanone in drinking water, or the 0.5 MTD concentration, or acidified water alone, the latter being the treatment for the controls. Three dose levels of cyclohexanone were used for female B6C3F1 mice, since the MTD (25,000 ppm) was so much higher than that for rats. In addition to the MTD and 0.5 MTD, another group of female mice was given the same concentration of cyclohexanone (6,500 ppm) as the rats.
Observations and examinations performed and frequency:
During the treatment, animals were weighed once a week for the first 4 months, then once every 2 weeks for 4 months, then once a month. Each cage of animals was inspected twice a day, and dead or moribund animals were removed for dissection and examination.
Sacrifice and pathology:
Three weeks after the end of scheduled treatment all animals were sacrificed and dissected. The following were fixed in Formalin, and slides were prepared and stained for histopathological examination: brain, pituitary gland, lymph nodes, spleen, thyroid gland, parathyroid glands, salivary glands, lung, trachea, heart, diaphragm, esophagus, stomach, gallbladder (in mice), duodenum, jejunum-ileum, large intestine, pancreas, kidneys, adrenal glands, liver, skin, gonads, urinary bladder, prostate, uterus, mammary gland, femur with marrow, and any lesions or tissue masses. In a very few cases, autolysis prevented satisfactory examination of some or all of the specimens.
Statistics:
Statistical methods used include life-table methods of Cox and Tarone.
Description (incidence):
No marked effects on survival were observed.
Description (incidence and severity):
Body weight retardation: A dose-related decrease in weight gain was evident in both male and female rats.
Description (incidence and severity):
Many neoplasms were seen in the treated animals; however, few neoplasms differed in incidences from the controls. See section "any other information on results"
Dose descriptor:
LOAEL
Effect level:
13 000 ppm
Sex:
male/female
Basis for effect level:
other: overall effects (based on about 18% decrease in body weight in males and drecreased survival in females)
Dose descriptor:
NOAEL
Effect level:
6 500 ppm
Sex:
male/female
Basis for effect level:
other: overall effects

Survivality of females at 13000 and 25000 ppm was poor. Fewer than 20% at 25000 ppm and approximately 50% at 13000 ppm were alive at 75 weeks. 80% of the males in the 13000 ppm group were alive at week 90.

Females: An increase in the incidence of malignant lymphoma was noted in the females treated with 6500 ppm but not at 13000 ppm.

Males: Increased hepatocellular neoplasms (combined adenomas and carcinomas) were increased at 6500 ppm but not at 13000 ppm.

The most significant histological findings in male mice involved proliferative lesions of liver and lung.

General Comments: Lack of a dose response for increased numbers of tumours already having a high background incidence prompted the authors to conclude that evidence of carcinogenicity was marginal and the effects, if any, were weak.

Table 1: Neoplasms in cyclohexanone-treated mice

Neoplasm Cyclohexanone doses
male mice female mice
0 6500 ppm 13000 ppm 0 6500 ppm 13000 ppm 25000 ppm
Lung: alveolar-bronchiolar adenoma or carcinoma 13/52 7/51 3/47 3/52 2/50 2/50 1/41
Lymphoma or leukemia 6/52 2/52 4/47 8/52 17/50 4/50 0/41
Liver: adenoma or carcinoma 16/52 25/51 13/46 3/52 6/50 3/50 2/41
Harderian gland: adenoma 0/52 4/52 0/47 0/52 1/50 1/50 0/41
Endpoint:
carcinogenicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
EPA OPP 83-5 (Combined Chronic Toxicity / Carcinogenicity)
GLP compliance:
not specified
Specific details on test material used for the study:
96% purity; 3% water, 1% unidentified compounds
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
F344 rats of both sexes (obtained from the Animal Production Facility of the NCI-Frederick Cancer Research Facility) were 7-8 weeks old at the beginning of treatment. The weight of the male rats averaged 162 g (139-180 g); that of the female rats, 126 g (107-143 g). In the chronic study, rats were housed 4 to a cage.
The study with both species (rats and mice) was conducted in a single animal holding room at 22-24°C in which there was frequent change of air (15 times per h). The animals were raised and housed behind a barrier, in a facility with clean-unclean corridors, the air pressure within the room being arranged so that air from the room would flow lo the unclean side. Mice were monitored for murine virus antibodies. The animals were fed Wayne Sterilizable Lab Meal ad libitum.
Controls received only acidified water and were kept in the same room. Each animal was weighed once a week and at the end of the study. Animals were sacrificed and dissected, the organs and tissues were fixed in Formalin, and slides were prepared for histopathological examination.
Route of administration:
oral: drinking water
Vehicle:
water
Details on exposure:
drinking water was acidified with HCl to prevent bacterial growth
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of the solution at intervals and after 28 days showed that there was no detectable decomposition during this time.
Duration of treatment / exposure:
104 weeks
Frequency of treatment:
continuously
Post exposure period:
none
Dose / conc.:
3 300 ppm
Remarks:
corresponding to 462 mg/kg bw/day (nominal in diet)
Dose / conc.:
6 500 ppm
Remarks:
corresponding to 910 mg/kg bw/day (nominal in diet)
No. of animals per sex per dose:
Each treatment group consisted of 50 or 52 male and 50 or 52 female rats.
Control animals:
yes, concurrent no treatment
Details on study design:
The rats and male mice were given either the MTD concentration of cyclohexanone in drinking water, or the 0.5 MTD concentration (defined during a 90day repeated dose toxicity study), or acidified water alone, the latter being the treatment for the controls.
Observations and examinations performed and frequency:
During the treatment, animals were weighed once a week for the first 4 months, then once every 2 weeks for 4 months, then once a month. Each cage of animals was inspected twice a day, and dead or moribund animals were removed for dissection and examination.
Sacrifice and pathology:
Three weeks after the end of scheduled treatment all animals were sacrificed and dissected. The following were fixed in Formalin, and slides were prepared and stained for histopathological examination: brain, pituitary gland, lymph nodes, spleen, thyroid gland, parathyroid glands, salivary glands, lung, trachea, heart, diaphragm, esophagus, stomach, gallbladder (in mice), duodenum, jejunum-ileum, large intestine, pancreas, kidneys, adrenal glands, liver, skin, gonads, urinary bladder, prostate, uterus, mammary gland, femur with marrow, and any lesions or tissue masses. In a very few cases, autolysis prevented satisfactory examination of some or all of the specimens.
Statistics:
Statistical methods used include life-table methods of Cox and Tarone.
Description (incidence):
No marked effects on survival were observed. At 6,500 ppm, more than 85% of male rats and female rats were alive at week 90, and 70% were alive at termination of the study. Survival of the 0.5 MTD-treated groups (3,300 ppm) was little different from the controls.
Description (incidence and severity):
Both male and female rats receiving 6,500 ppm of cyclohexanone for 2 years showed a considerably smaller weight gain throughout the experiment compared with the weight gain seen in controls. Even rats receiving 3,300 pprn failed to reach the weight of the untreated animals, although they gained more than the animals in the high dose group.
Description (incidence and severity):
There were no non-neoplastic lesions in the rats that differed significantly in incidence between treated and untreated animals that could be attributed to the treatment with cyclohexanone.
Description (incidence and severity):
At concentrations of 3,300 ppm, 13 % of the male rats had adrenocortical adenomas. In the male rats of the high dose group there was also an increase in follicular tumors of the thyroid (also see "Any other information on results")
Details on results:
Lack of a dose response for increased numbers of tumors already having a high background incidence prompted the authors to conclude that evidence of carcinogenicity was marginal and the effects, if any, were weak.

Dose descriptor:
LOAEL
Effect level:
3 300 ppm
Sex:
male/female
Basis for effect level:
other: overall effects (based on about 13% decrease in body weight)

Table 1: Neoplasms in cyclohexanone-treated F344 rats

Neoplasm Cyclohexanone doses
male rats female rats
0 3300 ppm 6500 ppm 0 3300 ppm 6500 ppm
Adrenal cortex: adenoma 1/52 7/52 1/51 8/52 4/51 4/52
Thyroid gland: follicular cell adenoma-carcinoma 1/52 0/51 6/51 0/52 1/52 1/52
Mammary gland: fibroadenoma 2/52 1/52 0/52 13/52 10/52 4/52
Uterus: endometrial stromal polyp       5/52 6/52 1/51
Liver:            
Carcinoma 2/52 0/52 0/51 0/52 0/52 0/52
Carcinoma plus neoplastic nodules 6/52 5/52 4/51 3/52 4/52 5/52
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
462 mg/kg bw/day
Study duration:
chronic
Species:
rat

Carcinogenicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Carcinogenicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the whole of information provided, classification according to Regulation (EC) 1272/2008 is not warranted.

Additional information