3,5,5-trimethylcyclohex-2-enone

Carcinogenicity

Description of key information

There was some evidence of carcinogenicity of the test substance in male rats (kidney tumors, preputial gland carcinomas). The kidney tumors can be attributed to an α2u-globulin associated mechanism. The observed nephropathy in male rats is therefore irrelevant to other species. As the preputium is only investigated histopathologically when gross lesions are found, neither true tumor incidences from this study nor from historical controls are available. Therefore, the higher incidence of preputial gland tumors in high dose male rats cannot be put into perspective. There was equivocal evidence of carcinogenicity for male mice (liver tumors, mesenchymal tumors of the integumentary system). There was no evidence of carcinogenicity of the test substance in female rats and mice.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Restrictions: no organ weight determination, no clinical biochemistry

Qualifier:

no guideline followed

Principles of method if other than guideline:

The test substance was administered to male and female F344/N rats by gavage in corn oil 5 days/week for 103 weeks. Body weights and clinical signs were recorded weekly during the first 13 weeks and then monthly. Animals were killed with carbon dioxide. Necropsies were performed on all animals, and tissues were examined for gross lesions and masses. Tissues were preserved in 10% neutral buffered formalin, embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined for non-neoplastic and neoplastic lesions.

GLP compliance:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Portage MI
- Age at study initiation: 6 - 8 weeks
- Weight at study initiation: not specified
- Fasting period before study: not specified
- Housing: 5 animals per cage
- Diet, ad libitum: NIH-07 pellets, Zeigler Brothers, Gardners PA
- Water, ad libitum: not further specified
- Acclimation period: 15 - 18 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 27°C, average 23°C
- Humidity (%): 29 - 74%, average 56%
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Dosing volume: 5 mL/kg bw

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

103 weeks

Frequency of treatment:

5 days/week

Post exposure period:

none

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Dose / conc.:

500 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

50

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS AND FREQUENCY
Clinical signs: weekly during the first 13 weeks and then monthly

Mortality: not specified

Body weight: weekly during the first 13 weeks and then monthly

Hematology: no

Urinalysis: no

Biochemistry: no

Sacrifice and pathology:

ORGANS EXAMINED AT NECROPSY (MACROSCOPIC AND MICROSCOPIC):
- Macroscopic: tissues were examined for gross lesions and masses
- Microscopic: gross lesions and tissue masses, skin, mammary gland, thymus, heart, lungs and bronchi, trachea, thyroid gland, parathyroids, esophagus, stomach, colon, small intestines, mesenteric lymph node, pancreas, spleen, liver, kidneys, adrenal glands, urinary bladder, prostate/testes or ovaries/uterus, brain, pituitary gland, eyes (if grossly abnormal), thoracic vertebrae, including bone marrow and spinal cord

Statistics:

- mortalities: life table analysis
- tumor incidence: life table analysis, incidental tumor test

Clinical signs:

no effects observed

Description (incidence and severity):

No compound-related clinical signs were observed.

Mortality:

mortality observed, treatment-related

Description (incidence):

Animals killed at termination
Males: 33 (control) / 33 (250 mg/kg) / 14 (500 mg/kg)
Females: 30 (control) / 23 (250 mg/kg) / 20 (500 mg/kg)

Non-accidental deaths:
Males: 13 (control) / 12 (250 mg/kg) / 30 (500 mg/kg)
Females: 19 (control) / 21 (250 mg/kg) / 16 (500 mg/kg)
500 mg/kg bw, m: increased mortality (after week 98)

Gavage-related trauma accounted for all accidental deaths.

The adjusted survival curves (accidental deaths censored) suggest that the test substance was associated with early deaths of several high dose male and female rats, and with a steep decline in survival of high dose male rats after about week 90.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights of high dose male rats averaged 5% less than controls, and body weights of female rats averaged 8% less than controls during the second year of the studies.

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

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Kidneys:
- Mineralization, primarily in the medullary collecting ducts, was increased in dosed male rats, and decreased in dosed female rats.
- The incidence of nephropathy was increased in dosed female rats.
These effects were not pronounced, however, and there was no evidence of renal injury in the prechronic studies, in which higher doses were given than in the 2-year study.

Adrenal cortex, m:
- fatty metamorphosis (7/50, 21/50, 26/50) (lesions in which adrenal cortical cells contained cytophalsmic vacuoles)

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Kidneys:
- In male rats variety of proliferative lessions in kidneys (hyperplasia, adenoma, and adenocarcinoma of the proximal tubule, and hyperplasia of the transitional cell epithelium in the renal pelvis).
The observed incidences of these lesions were relatively low in dosed male rats, but the overall dose-response trend was statistically significant (P < 0.05) when survival differences were taken into account.

Preputial gland, m:
- carcinoma (0/50, 0/50, 5/50) - Lesions were noted macroscopically and generally were greater than 1 cm.
Histopathology of preputial gland was only performed on high dose group.

Conclusion:
Some evidence of carcinogenicity is observed in male rats (renal tubular cell adenomas and adenocarcinomas, carcinomas of the preputial gland).
No evidence of carcinogenicity is observed in female rats.
The kidney tumors are probably attributed to an a2µ-globulin mechanism, see additional remarks (chapter 7.12).

Dose descriptor:

LOAEL

Effect level:

250 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: neoplastic

histopathology: non-neoplastic

Critical effects observed:

yes

Lowest effective dose / conc.:

250 mg/kg bw/day (actual dose received)

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Table 1: Survival of male and female rats

	Vehicle control	250 mg/kg	500 mg/kg
Animals initially in study (all groups)	50	50	50
Male rats Natural deaths before termination * Accidentally killed Killed at termination Survival P values **	13 4 33 <0.001	12 5 33 0.917	30 6 14 <0.001
Female rats Natural deaths before termination Accidentally killed Killed at termination Survival P values	19 1 30 0.748	21 6 23 0.537	16 14 20 0.886

* Includes animals that were killed in a moribund condition.

** The vehicle control column contains results of the life table trend test; the columns for dosed groups contain the life table exact pairwise comparisons with the vehicle controls.

Table 2: Renal lessions in male and female rats

	Vehicle control	250 mg/kg	500 mg/kg
Number examined in each group	50	50	50
Male rats Nephropathy Tubule mineralization Epithelial hyperplasia of the renal pelvis Tubular cell hyperplasia Tubular cell adenoma * Tubular cell adenocarcinoma *	49 1 0 0 0 0	47 31 5 1 0 3	46 20 5 4 2 1
Female rats Nephropathy Tubule mineralization Epithelial hyperplasia of the renal pelvis Tubular cell hyperplasia	21 10 0 0	39 4 0 0	32 2 1 1

* The incidence of tubular cell adenomas and adenocarcinomas (combined) showed a statistically significant (P < 0.05) dose-related increase.

Reference 2

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Restrictions: no organ weight determination, no clinical biochemistry

Qualifier:

no guideline followed

Principles of method if other than guideline:

The test substance was administered to male and female B6C3F1 mice by gavage in corn oil 5 days/week for 103 weeks. Body weights and clinical signs were recorded weekly during the first 13 weeks and then monthly. Animals were killed with carbon dioxide. Necropsies were performed on all animals, and tissues were examined for gross lesions and masses. Tissues were preserved in 10% neutral buffered formalin, embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined for non-neoplastic and neoplastic lesions.

GLP compliance:

no

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Portage MI
- Age at study initiation: 6 - 8 weeks
- Weight at study initiation: not specified
- Fasting period before study: not specified
- Housing: 5 animals per cage
- Diet, ad libitum: NIH-07 pellets, Zeigler Brothers, Gardners PA
- Water, ad libitum: not further specified
- Acclimation period: 15 - 18 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 27°C, average 23°C
- Humidity (%): 29 - 74%, average 56%
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Dosing volume: 10 mL/kg

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

103 weeks

Frequency of treatment:

5 days/week

Post exposure period:

none

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Dose / conc.:

500 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

50

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS AND FREQUENCY
Clinical signs: weekly during the first 13 weeks and then monthly

Mortality: not specified

Body weight: weekly during the first 13 weeks and then monthly

Hematology: no

Urinalysis: no

Biochemistry: no

Sacrifice and pathology:

ORGANS EXAMINED AT NECROPSY (MACROSCOPIC AND MICROSCOPIC):
- Macroscopic: tissues were examined for gross lesions and masses
- Microscopic: gross lesions and tissue masses, skin, mammary gland, thymus, heart, lungs and bronchi, trachea, thyroid gland, parathyroids, esophagus, stomach, colon, small intestines, mesenteric lymph node, pancreas, spleen, liver, kidneys, adrenal glands, urinary bladder, prostate/testes or ovaries/uterus, brain, pituitary gland, eyes (if grossly abnormal), thoracic vertebrae, including bone marrow and spinal cord

Statistics:

- mortalities: life table analysis
- tumor incidence: life table analysis, incidental tumor test

Clinical signs:

no effects observed

Description (incidence and severity):

No compound-related clinical signs were observed.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Animals killed at termination:
Males: 16 (control) / 16 (250 mg/kg) / 19 (500 mg/kg)
Females: 26 (control) / 35 (250 mg/kg) / 34 (500 mg/kg)

Non-accidental deaths:
Males: 28 (control) / 34 (250 mg/kg) / 29 (500 mg/kg)
Females: 23 (control) / 14 (250 mg/kg) / 11 (500 mg/kg)

The test substance did not adversely affect survival of male or female mice, although several dosed mice died early in the study. Adjusted survival among dosed females was somewhat better than for vehicle controls. Fighting among the group housed male mice may have contributed to the poor survival.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weights of high dose females averaged about 5% less than controls during the second year of the study. No effect on body weight was noted in any other group of dosed mice.

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

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Several tumor types were increased marginally in dosed male mice:
- Hepatocellular neoplasms (adenomas and carcinomas) occurred with a positive trend (P < 0.05) (control, 18/48; low dose, 18/50;
high dose, 29/50). The incidence of liver neoplasia in female mice was 4/5 0, 6/50, 8/50.
- Mesenchymal tumors of the integumentum consisting primarily of subcutaneous fibrosarcomas, were observed at increased incidence in high dose male mice (control, 6/48; low dose 8/50; high dose, 14/50) (P < 0.05). These tumors often appeared as firm nodular masses, sometimes associated with ulceration of the overlying skin.
- The incidence of lymphomas or leukemias (8/48, 19/50, 5/50) (primarily lymphomas, one leukemia was noted in a control mouse, and none in dosed male mice) was increased marginally in low dose male mice, but this inerease was not statistically significant by life table analysis.

Dose descriptor:

LOAEL

Effect level:

250 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: neoplastic

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

Critical effects observed:

no

Table 1: Survival of male and female mice

	Vehicle control	250 mg/kg	500 mg/kg
Animals initially in study (all groups)	50	50	50
Male mice Natural deaths before termination Accidentally killed Animals missing Killed at termination Survival P values	28 5 1 16 0.699	34 0 0 16 0.844	29 2 0 19 0.780
Female mice Natural deaths before termination Accidentally killed Killed at termination Survival P values	23 1 26 0.045	14 1 35 0.086	11 5 34 0.077

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

250 mg/kg bw/day

Study duration:

chronic

Species:

other: rat and mouse

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

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is considered to be classified for carcinogenicity cat. 2, H351 under Regulation (EC) No. 1272/2008, as amended for the 14th time in Regulation (EU) 2020/217.

Additional information

In a 2-year study (Bucher et al., 1986) Fischer-344 rats (50 animals/dose/sex) were exposed by gavage to daily doses (5 days/week) of 0, 250, or 500 mg/kg isophorone in corn oil. The overall survival rate was low (males 33/50, 33/50, 14/50; females 30/50, 23/50, 20/50). Increased mortality was observed after week 98 in males in the 500 mg/kg bw dose group. Compound related clinical signs were not observed. Throughout the study, the mean body weights of high dose males averaged 5 % lower than vehicle controls. During the second year, the mean body weights of high dose females averaged 8 % lower than vehicle controls.

Dosed males showed proliferative lesions of the kidney (tubular cell hyperplasia: 0/50, 1/50, 4/50; tubular cell adenoma: 0/50, 0/50, 2/50; tubular cell adenocarcinoma: 0/50, 3/50, 1/50; epithelial hyperplasia of the renal pelvis: 0/50, 5/50, 5/50) and an increased mineralization of the medullary collecting ducts (1/50, 31/50, 20/50). Low dose males revealed more severe nephropathy than is commonly observed in aging F344 rats. Female rats only showed a statistically significant increase in nephropathy (21/50, 39/50, 32/50) with no further findings in the kidneys.

Carcinomas of the preputial gland were increased in high dose males only (0/50, 0/50, 5/50). The preputium is only investigated histopathologically when gross lesions are found, therefore neither true tumor incidences from this study nor from historical controls are available.

In a parallel 2-year gavage study with B6C3F1 mice (50 animals/dose/sex) animals were dosed daily (5 days/week) with 0, 250, or 500 mg/kg isophorone in corn oil. The survival of male mice was low (16/50, 16/50, 19/50) in contrast to female mice, where there was a trend towards increased survival of dosed animals relative to the controls (26/50, 35/50, 34/50). In high dose males, there was an increased incidence of hepatocellular adenomas and carcinomas (18/48, 18/50, 29/50). Mesenchymal tumors of the integumentary system (6/48, 8/50, 14/50) were increased in high dose males. An increased incidence of lymphomas or leukemias was noted in low dose males only (8/48, 19/50, 5/50). These results were interpreted as chemically related marginal increases in number of neoplasms (equivocal evidence).

 

Investigations on α2u-globulin induced nephropathy:

Various chemicals are known to induce nephropathy in male rats only. Only 60 % of α2u-globulin is reabsorbed in the kidney of male rats. 40 % of this low molecular weight protein remains in the filtrate and is excreted to the urine. The amount of α2u-globulin in female rats is 120 times lower and the protein is nearly absent in mice and in men. Chemicals that induce α2u-globulin nephropathy bind to the protein in the liver of the animals; these conjugates are difficult to hydrolyse and induce the formation of hyaline droplets which accumulate in the tubules (Charbonneau and Swenberg, 1988; Swenberg et al., 1989).

Isophorone as well as the proposed metabolites isophorol and dihydroisophorone form protein complexes with α2u-globulin in vivo, (SDS-page and immunoblotting after 14 d isophorone administration; GC/MS determination of isolated protein complexes) (Saito et al., 1992; Strasser et al., 1988). The degradation of α2u-globulin via lysosomal proteinases was decreased by 33 % (Lehman-McKeeman et al., 1990). In a further experiment with male NCI-Black-Reiter rats (strain is unable to synthesize the hepatic form of the low molecular weight protein α2u -globulin) isophorone administration, which clearly induced kidney lesions in male F344 rats, failed to induce nephropathy, α2u-globulin or the formation of hyaline droplets (Dietrich and Swenberg, 1990). It is thus concluded that the observed nephropathy in male rats is caused via the α2u-globulin mechanism and therefore irrelevant to other species.

Charbonneau M and Swenberg JA (1988). Studies on the Biochemical Mechanism of α2u-Globulin Nephropathy in Rats. Chemical Industry Institute of Toxicology Vol. 8, No. 6

Swenberg JA, Short B, Borghoff S, Strasser J and Charbonneau M (1989). The Comparative Pathology of α2u-Globulin Nephropathy. Toxicol Appl Pharmacol. 1989 Jan; 97 (1):35–46

Strasser J, Charbonneau M, Borghoff SJ, Turner MJ, Swenberg JA (1988). Renal protein droplet formation in male Fischer 344 rats after isophorone (IPH) treatment. Toxicologist 8:136 (abstract)

Lehman-McKeeman LD, Rivera-Torres MI, Caudill D. (1990). Lysosomal degradation of α2u-globulin and α2u-globulin-xenobiotic conjugates. Toxicol Appl Pharmacol. May 1990; 103 (3):539–548