3,5,5-trimethylcyclohex-2-enone

Carcinogenicity

Description of key information

There was some evidence of carcinogenicity of isophorone in male rats (kidney tumors, preputial gland carcinomas) (NTP, 1986; rats). The kidney tumors can be attributed to an alpha2µ-globin 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 integumentary system) (NTP, 1986; mice).
There was no evidence of carcinogenicity of isohphorone 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

Study period:

1980-01-31 to 1982-01-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study

Principles of method if other than guideline:

method: NTP-Carcinogenicity Study; comparable to guideline study

GLP compliance:

no

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ORGANISMS
-  Number of animals: 50 per dose and sex
- Source: Charles River Breeding, Laboratories
- Age at study initiation: 6-8 weeks
- Age when killed: 110-113 weeks
- Diet: ad libitum. Purina Lab Chow
- Water: ad libitum
- Acclimation period: 15 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 27 °C, average: 23 °C
- Humidity (%): 29 - 74 %, average: 56 %
- Air changes (per hr): 10 - 15 times per hour
- Photoperiod (hrs dark / hrs light): 12 hours/day
- Housing: five per cage in polycarbonate cages

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

ADMINISTRATION / EXPOSURE
- Vehicle: corn oil
- Concentration in vehicle: 25 and 50 mg/ml, respectively
- Total volume applied: 10 ml/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

All chemical/vehicle analyses were performed by gas chromatography
Isophorone in corn-oil was found to be stable for 7 days at room temperature
storage conditions: 2° - 8° C

Duration of treatment / exposure:

103 weeks

Frequency of treatment:

5 days/week

Post exposure period:

none

Remarks:

Doses / Concentrations:
250 and 500 mg/kg bw d
Basis:
nominal in diet

No. of animals per sex per dose:

50

Control animals:

yes, concurrent vehicle

Details on study design:

Post-exposure period: none

Positive control:

none

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS AND FREQUENCY
- Body weight: weekly for first 13 weeks, then monthly
- Clinical signs: weekly for first 13 weeks, then monthly
- Mortality: twice daily
- Macroscopic examination: twice daily
- Moribund animals were killed, as were animals that survived to the end of the study; a necropsy was performed on all animals, including those found dead unless they were excessively autolyzed or cannibalized.

Sacrifice and pathology:

ORGANS EXAMINED AT NECROPSY (MACROSCOPIC AND MICROSCOPIC): 
- Macroscopic: no details reported
- 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, gallblader, 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

Other examinations:

none

Statistics:

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

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

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

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

effects observed, treatment-related

Details on results:

Result (carcinogenicity): ambiguous (equivocal evidence of carcinogenicity in male mice, no evidence in females)

MORTALITY AND TIME TO DEATH: 
The overall survival rate was:
Males:   13 (control) / 13 (250 mg/kg) / 18 (500 mg/kg)
Females: 24 (control) / 33 (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)
>= 250 mg/kg bw f: decrease mortality (non-accidental kills:
23/50, 14/50, 11/50). 
Further deaths are documented to be due to gavage accidents.

CLINICAL SIGNS: 
No compound-related clinical signs were observed.

BODY WEIGHT GAIN:  500 mg/kg bw f: body weight 5% lower (during the 2nd year)

HISTOPATHOLOGY: 
Liver, m:
Hepatocellular adenoma: 6/48, 7/50, 13/50*
Hepatocellular carcinoma: 14/48, 13/50, 22/50*
Hepatocellular adenoma or carcinoma: 18/48, 18/50, 29/50*
Coagulative necrosis: 3/48, 10/50, 11/50
Hepatocytomegaly: 23/48, 39/50, 37/50
Liver, f: Hepatocellular adenoma or carcinoma: 4/50, 6/50, 8/50,
Skin, m: Fibroma, Sarcoma, Fibrosarcoma or neurofibrosarcoma: 6/48, 8/50, 14/50 (P  = 0,050)
Skin, f: one sarcoma in low dose, one fibrosarcoma in high dose
Hematopoietic system, 
m: Lymphoma or Leukemia: 8/48, 18/50*, 5/50
f: Lymphoma or Leukemia: no significantly different from vehicle control
Forestomach: Hyperkeratosis was observed at increased incidences in dosed male and high dose female
m: 0/47, 5/49, 4/49; f: 1/50, 0/50, 5/49
Lung, m: Alveolar/bronchiolar adenoma or carcinoma: 7/47, 1/50, 3/50  (significantly negative trend)
Kidney, m: Chronic focal inflammation: 7/48, 18/50, 21/50
Nephropathy: m: 16/48, 15/50, 9/50 f: 13/50, 8/50, 2/50, incidence of nephropathy in dosed mice of each sex were lower than those in the
vehicle
Pituitary gland, f:
Hyperplasia: 5/47, 7/41, 13/44
Adenoma or adenocarcinoma: 16/47, 13/41, 4/44 (significant negative trend)

* P < 0.05

Conclusion: Equivocal evidence of carcinogenicity in male mice (increase  in hepatocellular und integumentary tumors). No evidence of  
carcinogenicity is observed in female rats.

Relevance of carcinogenic effects / potential:

male mice, hepatocellular and integumentary tumors: equivocal evidence of carcinogenicity

Dose descriptor:

LOAEL

Effect level:

>= 250 mg/kg bw/day (nominal)

Sex:

male

Basis for effect level:

other: increase in malignant lymphomas (hematopoietic system)

Remarks on result:

other: Effect type: carcinogenicity (migrated information)

no remarks

Conclusions:

Under the conditions of this 2-year gavage study, there was equivocal evidence of carcinogenicity of isophorone in B6C3F1-mice as shown by an
increased incidence of hepatocellular adenomas or carcinomas (combined) and of mesenchymal tumours in the integumentary system in animals
given 500 mg/kg per day and by an increase in malignant lymphomas in animals given 250 mg/kg day. There was no evidence of carcinogenicity of
isophorone in female B6C3F1 mice given 250 or 500 mg/kg per day.

Executive summary:

In this NTP 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 (13/50, 13/50, 18/50) in contrast to female mice, where there was a trend towards increased survival of dosed animals relative to the controls (24/50, 33/50, 34/50). Mean body weights of dosed and vehicle control male mice were comparable throughout most of the study. The mean body weights of high dose and vehicle control female mice were comparable for the 1st year of the study. During the 2nd year of the study, mean body weights of high dose female mice averaged about 5 % lower than those of the vehicle controls. No compound-related clinical signs were observed.

In high dose males, there was an increased incidence of hepatocellular adenomas and carcinomas (18/48, 18/50, 20/50). Increased incidences of coagulative liver necrosis (3/48, 10/50, 11/50) and hepatocytomegaly (23/48, 39/50, 37/50) were found in low and high dose males.

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 male only (8/48, 18/50, 5/50). These results were interpreted as chemically related marginal increases in number of neoplasms (equivocal evidence).

Compound related non-neoplastic or neoplastic lesions associated with isophorone exposure were not seen in female mice.

Therefore, under the conditions of this 2 -year gavage study, there was equivocal evidence of carcinogenicity of isophorone in male B6C3F1 mice as shown by an increased incidence of hepatocellular adenomas or carcinomas (combined) and of mesenchymal tumours in the integumentary system in animals given 500 mg/kg per day and by an increase in malignant lymphomas in animals given 250 mg/kg per day. There was no evidence of carcinogenicicty in female B6C3F1 mice.

Reference 2

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1980-01-31 to 1982-01-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study

Principles of method if other than guideline:

Method: NTP-Carcinogenicity Study; comparable to guideline study

GLP compliance:

no

Species:

rat

Strain:

other: Fischer 344/N

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ORGANISMS
- Number of animals: 50 per dose group and sex
- Source: Charles River Breeding Laboratories
- Age at study initiation: 6 - 7 weeks
- Age when killed: 111 - 112 weeks
- Diet: ad libitum. NIH 07 pellets
- Water: ad libitum
- Acclimation period: 15 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 27 °C, average 23°C
- Humidity (%): 29 % - 74 %, average 56 %
- Air changes (per hr): 10 - 15 times per hour
- Photoperiod (hrs dark / hrs light): 12 hours/day
- Housing: five per cage in polycarbonate cages

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

ADMINISTRATION / EXPOSURE
- Vehicle: corn oil
- Concentration in vehicle: 50 and 100 mg/ml, respectively
- Total volume applied: 5 ml/kg
- Isophorone was added to corn oil in a graduated cylinder. Dose mixtures were prepared by further diluting this stock solution with corn oil to the
appropriate concentrations.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- All chemical/vehicle analyses were performed by gas chromatography
- Isophorone in corn-oil was found to be stable for 7 days at room temperature
- storage conditions: 2° - 8° C

Duration of treatment / exposure:

103 weeks

Frequency of treatment:

5 days/week

Post exposure period:

none

Remarks:

Doses / Concentrations:
250 and 500 mg/kg bw d
Basis:
nominal in diet

No. of animals per sex per dose:

50

Control animals:

yes, concurrent vehicle

Details on study design:

Post-exposure period: none

Positive control:

none

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS AND FREQUENCY
- Body weight: weekly for first 13 weeks, then monthly; mean body weights were calculated for each group
- Clinical signs: weekly for first 13 weeks, then monthly
- Mortality: twice daily
- Macroscopic examination: twice daily
- Moribund animals were killed, as were animals that survived to the end of the study; a necropsy was performed on all animals, including those found dead unless they were excessively autolyzed or cannibalized.

Sacrifice and pathology:

ORGANS EXAMINED AT NECROPSY (MACROSCOPIC AND MICROSCOPIC): 
- Macroscopic: no details reported
- 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

Other examinations:

none

Statistics:

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

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

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

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

effects observed, treatment-related

Details on results:

Result (carcinogenicity): ambiguous (some evidence of carcinogenicity in male rats, no evidence in females)

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.

MORTALITY AND TIME TO DEATH: 
The overall survival rate was low:
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: survival of the high dose group of male was signifcantly lower than that of the vehicle control group after week 96 
Gavage errors accounted for all of the 36 accidental death of male and female rats.
Deaths related to gavage error increased with dose in females.

BODY WEIGHT GAIN: 
500 mg/kg bw, m: body weight was 5% lower (after week 1)
500 mg/kg bw, f: body weight was 8% lower (after week 43)

CLINICAL SIGNS:  No compound-related clinical signs were observed.

HISTOPATHOLOGY: 
Kidneys: m (controls, 250, 500 mg/kg bw):
- 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*)
- tubule mineralization (1/50, 31/50, 20/50)
- Nephropathy (49/50, 47/50, 46/50) 
- with higher severity in low dose males
 Kidney, f:
- Nephropathy (21/50, 39/50*, 32/50*) No further compound-related findings in kidneys of females.
Adrenal cortex: m
- fatty metamorphosis (7/50, 21/50, 26/50) (lesions in which adrenal  cortical cells contained cytophalsmic vacuoles, small vacuoles often contained eosinophilic fibreillar material, this lesion was most frequently seen in zona fasciculata)
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.
Clitoral gland, f: 
- adenomas (0/50, 2/50, 0/50) 
- These lesions were  histogenically  related to the preputial gland carcinomas observed in male rats,  providing some support for an association of 
isophorone exposure with  this tumor type.
Pancreas: Incidences of hyperplasia were similar in dosed and vehicle control groups. Results of statistical analysis of the incidence of male rats with
hyperplasia or adenomas were similar to those of male rats with adenomas. Acinar cell adenomas occurred in male rats with significant positive trend by the life table test. The incidence in the high dose group was significant greater than that in the vehicle controls only by the life table test.
Anterior Pituitary, f:
- focal hyperplasia (3/49, 6/48*, 13/47*)
But incidence of adenomas occurred with a negative trend in female rats  (21/49, 17/48, 12/47)

* statistically significant

Relevance of carcinogenic effects / potential:

male rats, carcinomas of the preputial gland: relevant carcinogenic effect

Dose descriptor:

LOAEL

Effect level:

>= 500 mg/kg bw/day (nominal)

Sex:

male

Basis for effect level:

other: Carcinomas of the preputial gland: lesions were noted macroscopically and generally were greater than 1 cm. Histopathology of preputial gland was only performed on high dose group

Remarks on result:

other: Effect type: carcinogenicity (migrated information)

Dose descriptor:

LOAEL

Effect level:

>= 250 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: female: nephropathy male: renal tubular cell hyperplasia

Remarks on result:

other: Effect type: toxicity (migrated information)

no remarks

Conclusions:

Under the conditions of this 2-year gavage study, there was some evidence of carcinogenicity of isophorone in male F-344/N rats as shown by the
occurrence of renal tubular cell adenomas and adenocarcinomas in animals given 250 or 500 mg/kg per day. It is concluded that the observed
nephropathy in male rats is caused via the alpha2u-globulin mechanism and therefore irrelevant to other species. Carcinomas of the perputial gland were also observed at increased incidence in male rats given 500 mg/kg. There was no evidence of carcinogenicity in female F-344/N rats given 250 or 500 mg/kg per day.

Executive summary:

In this NTP 2 -year study Fischer-344/N 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; female 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 the vehicle control.

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/N 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 male 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. As also two clitoral gland tumors were found in low dose females, a treatment related effect cannot be discounted.

Therefore, under the conditions of this 2-year gavage study, there was some evidence of carcinogenicity of isophorone in male F-344/N rats as shown by the occurrence of renal tubular cell adenomas and adenocarcinomas. It is concluded that the observed nephropathy in male rats is caused via the alpha2u-globulin mechanism and therefore irrelevant to other species. Carcinomas of the perputial gland were also observed at increased incidence in male rats given 500 mg/kg. There was no evidence of carcinogenicity in female F-344/N rats.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Study duration:

chronic

Species:

other: rats and mice

Quality of whole database:

Klimisch 1 (reliable without restrictions)

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

According to the available carcinogenicity data isphorone shows a limited evidence for a carcinogenic effect (CLP Regulation 1272/2008: Cat. 2, H351 "Suspected of causing cancer").

Additional information

In a NTP 2 -year study with Fischer-344/N rats (50 animals/dose/sex) animals 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; female 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 the vehicle control.

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/N 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 male 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. As also two clitoral gland tumors were found in low dose females, a treatment related effect cannot be discounted.

Therefore, under the conditions of this 2-year gavage study, there was some evidence of carcinogenicity of isophorone in male F-344/N rats as shown by the occurrence of renal tubular cell adenomas and adenocarcinomas. It is concluded that the observed nephropathy in male rats is caused via the alpha2u-globulin mechanism and therefore irrelevant to other species. Carcinomas of the perputial gland were also observed at increased incidence in male rats given 500 mg/kg. There was no evidence of carcinogenicity in female F-344/N rats.

In the same NTP 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 (13/50, 13/50, 18/50) in contrast to female mice, where there was a trend towards increased survival of dosed animals relative to the controls (24/50, 33/50, 34/50). Mean body weights of dosed and vehicle control male mice were comparable throughout most of the study. The mean body weights of high dose and vehicle control female mice were comparable for the 1st year of the study. During the 2nd year of the study, mean body weights of high dose female mice averaged about 5 % lower than those of the vehicle controls. No compound-related clinical signs were observed.

In high dose males, there was an increased incidence of hepatocellular adenomas and carcinomas (18/48, 18/50, 20/50). Increased incidences of coagulative liver necrosis (3/48, 10/50, 11/50) and hepatocytomegaly (23/48, 39/50, 37/50) were found in low and high dose males.

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 male only (8/48, 18/50, 5/50). These results were interpreted as chemically related marginal increases in number of neoplasms (equivocal evidence).

Compound related non-neoplastic or neoplastic lesions associated with isophorone exposure were not seen in female mice.

Therefore, under the conditions of this 2 -year gavage study, there was equivocal evidence of carcinogenicity of isophorone in male B6C3F1 mice as shown by an increased incidence of hepatocellular adenomas or carcinomas (combined) and of mesenchymal tumours in the integumentary system in animals given 500 mg/kg per day and by an increase in malignant lymphomas in animals given 250 mg/kg per day. There was no evidence of carcinogenicicty in female B6C3F1 mice.

Investigations on alpha2µ-globin induced nephropathy (see chapter 7.12 of IUCLID 5)

Various chemicals are known to induce nephropathy in male rats only. Only 60 % of alpha2µ-globin is resorbed 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 alpha2µ-globin in female rats is 120 times lower and the protein is nearly absent in mice and in men. Chemicals that induce alpha2µ-globin nephropathy bind to the protein in the liver of the animals; these conjungates 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 alpha2µ-globin in vivo (Saito et al., 1992). The degradation of alpha2µ-globin via lyosomal proteinases was decreased by 33 % (Lehman-McKeeman et al., 1990). In a further experiment with male NCI-Black-Reiter rats isophorone administration, which clearly induced kidney lesions in male F344 rats, failed to induce nephropathy, alpha2µ or the formation of hyaline droplets (Dietrich and Swenberg, 1991).

It is thus concluded that the observed nephropathy in male rats is caused via the alpha2µ-globin mechanism and therefore irrelevant to other species.

Justification for selection of carcinogenicity via oral route endpoint:
No study is selected, since all studies are relevant for assessment. There was some evidence of carcinogenicity of isophorone in male rats (kidney tumors, preputial gland carcinomas) (NTP, 1986; rats). The kidney tumors can be attributed to an alpha2µ-globin 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 prespective. There was equivocal evidence of carcinogenicity for male mice (liver tumors, mesenchymal tumors of integumentary system) (NTP, 1986; mice). There was no evidence of carcinogenicity of isohphorone in female rats and mice.