Cyclohexane, 1-methyl-4-(1-methylethyl)-, tetradehydro deriv.

Carcinogenicity

Description of key information

Two year carcinogenicity studies were available in rats and mice with d-limonene. In rats,  hyperplasia and adenomas were seen in male rats associated with cytoplasmic alfa 2µ-globulin granules in the renal tubuli. In mice, no hyperplasia or tumours were observed. The findings in male rats have meanwhile been accepted not to be relevant for humans. 

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: oral

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Based on a read across evaluation, the registered substance has a comparable physicochemical and toxicological profile to d-limonene. The study was conducted according to valid methods, however standards were slightly different at time of testing. The study can be considered as relevant, reliable and adequate for classification.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Deviations:

yes

Principles of method if other than guideline:

Dosing 5 days/week instead of 7 days/week; food consumption and clinical biochemical tests not followed.

GLP compliance:

yes

Species:

rat

Strain:

other: F344/N

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Portage, MI (produced under strict barrier conditions). Breeding stock for the foundation colonies at the production facility originated at the National Institutes of Health Repository.
- Age at study initiation: Shipped to the study laboratory at 4 to 5-week-old ; placed on study at 7-8 weeks of age.
- Weight at study initiation: Males: 183-187 g; females: 132-133 g
- Housing: Animals were housed five per cage. Cages were not rotated until the last 10 weeks of the studies.
- Diet: Ad libitum; NIH 07 Rat and Mouse Ration (Zeigler Bros., Inc., Gardners, PA, USA).
- Water: Ad libitum; Automatic watering system (Edstrom Industries, Waterford, USA).
- Acclimation period: The animals were quarantined at the study facility for 19 days. Thereafter, a complete necropsy was performed on five animals of each sex to assess their health status. The rats were placed on study at 7-8 weeks of age.

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 66-84 °F
- Humidity (%): 20-78%
- Air changes (per hour): 12-15/hour
- Photoperiod (hours dark / hours light): 12 hours dark / 12 hours light

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The appropriate amounts of d-limonene and corn oil were mixed to give the desired concentrations.

VEHICLE
-Amount of vehicle (gavage): 5 mL/kg bw
- Justification for use and choice of vehicle (if other than water): The stability of D-Limonene at 40000 ppm in feed was determined by extraction with methanol followed by gas chromatographic analysis of the extract with a 6-foot 3% OV-17 glass column, a nitrogen carrier at a flow rate of 30 mL/minute, and a flame ionization detector. Formulated diets containing d-limonene were not stable when stored for 2 weeks at temperatures above -20°C. Because the d-limonene/feed blends were determined to be unstable, corn oil was chosen as the vehicle for the gavage studies of D-Limonene.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability of d-limonene in corn oil was evaluated at the study and analytical chemistry laboratories after extraction of dose mixtures with methanol and quantitation by gas chromatography with a 3% OV-17 column, a helium or nitrogen carrier at a flow rate of 30 mL/minute, and flame ionization detection. At the analytical chemistry laboratory, d-limonene in corn oil (40 mg/mL) was found to be stable for up to 7 days at room temperature. Stability study results at the study laboratory showed that d-limonene in corn oil (12.5 and 240 mg/mL) was stable for at least 14 days at room temperature. In the 13-week studies, D-Limonene/corn oil mixtures were stored at room temperature for no more than 15 days. In the 2-year studies, the dose mixtures were stored at room temperature for no longer than 2 weeks.
Periodic analysis for d-limonene in dose preparations by the same methanol extraction and gas chromatographic quantitation step was performed by the study and analytical chemistry laboratories to determine if the dose mixtures contained the correct concentrations of D-Limonene. During the 2-year studies, the dose preparations were analyzed approximately every 8 weeks with concentrations varying from 87% to 110% of the target value. Because 85/91 dose mixtures analyzed in the 2-year studies were within 10% of the target concentrations, it is estimated that dose mixtures were prepared within specifications 93% of the time. Referee analyses were performed periodically by the analytical chemistry laboratory. Good agreement was generally found between the study and the analytical chemistry laboratories.

Duration of treatment / exposure:

103 weeks

Frequency of treatment:

5 days per week

Post exposure period:

1 week in males; 1-2 weeks in females

Remarks:

Doses / Concentrations:
male rats: 0, 75 and 150 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
female rats: 0, 300 and 600 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

50

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses were selected based on compound-related, potentially life-threatening kidney lesions observed in males at 300 mg/kg and higher and on the large number of deaths of female rats at 2400 mg/kg in the 13 weeks oral gavage study.
- Rationale for animal assignment: Random

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed two times per day. Animals found moribund and those surviving to the end of the studies were humanely killed.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded once per week for the first 12 weeks of the studies and once per month thereafter.
- Mean body weights were calculated for each group.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- A necropsy was performed on all animals including those found dead, unless they were excessively autolyzed or missing. Thus, the number of animals from which particular organs or tissues were examined microscopically varies and is not necessarily equal to the number of animals that were placed on study. During necropsy, all organs and tissues were examined for grossly visible lesions.

HISTOPATHOLOGY: Yes
- Tissues were preserved in 10% neutral buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Histopathologic examination of tissues was performed according to an "inverse pyramid" design (McConnell, 1983a,b). That is, complete histopathologic examinations (Table 7) were performed on all high dose and vehicle control animals and on low dose animals dying before the end of the study. In addition, histopathologic examinations were performed on all grossly visible lesions in all dose groups. Potential target organs for chemically related neoplastic and non-neoplastic effects were identified from the short-term studies or the literature and were determined by examination of the pathology data; these target organs/tissues in the lower dose group were examined histopathologically. If mortality in the highest dose group exceeded that in the vehicle control group by 15%, complete histopathologic examinations were performed on all animals in the second highest dose group in addition to those in the high dose group.
- When the pathology evaluation was completed by the laboratory pathologist and the pathology data entered into the Carcinogenesis Bioassay Data System, the slides, paraffin blocks, and residual formalin-fixed tissues were sent to the NTP Archives.The slides, blocks, and residual wet tissues were audited for accuracy of labelling and animal identification and for thoroughness of tissue trimming. The slides, individual animal necropsy records, and pathology tables were sent to an independent pathology quality assessment laboratory. The individual animal records and pathology tables were compared for accuracy, slides and tissue counts were verified, and histotechnique was evaluated. All tissues with a tumor diagnosis, all potential target tissues, and all tissues from a randomly selected 10% of the animals were re-evaluated microscopically by a quality assessment pathologist. Non-neoplastic lesions were evaluated for accuracy and consistency of diagnosis only in the potential target organs, in the randomly selected 10% of animals, and in tissues with unusual incidence patterns or trends. Tissues are generally not evaluated in a "blinded" fashion (i.e., without knowledge of dose group) unless the lesions in question are subtle.
- The quality assessment report and slides were submitted to a Pathology Working Group (PWG) Chairperson, who reviewed microscopically all potential target tissues and any other tissues for which there was a disagreement in diagnosis between the laboratory and quality assessment pathologists. Representative examples of potential chemical-related non-neoplastic lesions and neoplasms and examples of disagreements in diagnosis between the laboratory and quality assessment pathologists were shown to the PWG. The final pathology data represent a consensus of contractor pathologists and the NTP Pathology Working Group. For subsequent analysis of pathology data, the diagnosed lesions for each tissue type are combined according to the guidelines of McConnell et al. (1986).

Statistics:

- Survival Analyses: statistical analyses for possible dose-related effects on survival used Cox’s (1972) method for testing two groups for equality and Tarone’s (1975) life table test to identify dose-related trends.
- Analysis of Tumor Incidence: In addition to incidental tumor analysis, alternative methods of statistical analysis were used, and the results of these tests are summarized in the appendixes. These include the life table test (Cox, 1972;Tarone, 1975),appropriate for rapidly lethal tumors, and the Fisher exact test and the Cochran-Armitage trend test (Armitage, 1971;Gart et al., 1979), procedures based on the overall proportion of tumor-bearing animals.
Tests of significance include pairwise comparisons of each dosed group with vehicle controls and a test for an overall dose-response trend. Continuity-corrected tests were used in the analysis of tumor incidence, and reported P values are one-sided. The procedures described above also were used to evaluate selected non-neoplastic lesions. (For further discussion of these statistical methods, see Haseman, 1984.)

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Decrease of 4-7% versus vehicle controls: males from week 2 to the end of the studies; females from week 28 to the end of the study

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Kidney: The administration of d-limonene to male rats was associated with dose-related increased incidences of mineralization and epithelial hyperplasia (Table 13).

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Kidney: The administration of d-limonene to male rats was associated with dose-related increased incidences of mineralization and epithelial hyperplasia (Table 13).

Details on results:

CLINICAL SIGNS AND MORTALITY
- No compound­related clinical signs were observed during the 2-year studies.
- Survival of the high-dose male group was significantly greater than that of the vehicle controls after week 81.
- Survival of the high-dose female group was significantly lower than that of the vehicle controls after week 39.
- Survival at week 104-male: vehicle control, 29/50; low dose, 33/50; high dose, 40/50; female: 42/50; 40/50; 26/50.

BODY WEIGHT AND WEIGHT GAIN
- Mean body weights of high dose male rats (150 mg/kg bw) were generally 4%-7% lower than those of the vehicle controls from week 2 to the end of the study.
- Mean body weights of high dose female rats (600 mg/kg bw) were generally 4%-7% lower than those of the vehicle controls from week 28 to the end of the study.

HISTOPATHOLOGY
Kidney:
- Dose-related increased incidences of mineralization and epithelial hyperplasia. These lesions consisted of linear deposits of mineral in the medulla (renal papilla) and focal hyperplasia of the transitional epithelium overlying the papilla. The hyperplasia was often located near the fornices of the renal pelvis and was sometimes bilateral. There was also a dose-related increased severity of spontaneous nephropathy in dosed male rats. Nephropathy is an age-related disease characterized by degeneration and atrophy of the tubular epithelium, dilatation of tubules with formation of hyalin and granular casts, regeneration of tubular epithelium, glomerulosclerosis, and interstitial inflammation and fibrosis.
- Tubular cell hyperplasia and neoplasia were also observed at increased incidences in dosed male rats. Tubular cell adenomas and tubular cell adenomas or adenocarcinomas (combined) in male rats occurred with significant positive trends; the incidences of tubular cell adenomas in high dose male rats and of tubular cell adenomas or adenocarcinomas (combined) in dosed male rats were significantly greater than those in vehicle controls. These rare neoplasms were not observed in vehicle control male rats or in dosed or vehicle control female rats.
- No compound- related lesions of the uterus, testis, hematopoietic system, skin, or eye were noted in any of the rats dosed with D-limonene.

OTHER FINDINGS
In the 21-day studies conducted after the 2-year studies, microscopic examination of paraffin-embedded sections of kidney stained with hematoxylin and eosin showed no visible differences between dosed and vehicle control male and female rats. However, in plastic-embedded sections of kidney stained with Lee’s methylene blue basic fuchsin, differences in the distribution, amount, and shape of intracytoplasmic granules in the proximal tubules of dosed and vehicle control males were detected. In vehicle control male rats, focal clusters of proximal convoluted tubules contained varying numbers of round, blue-to-purple granules of varying size. In dosed males, the tubular cells containing the intracytoplasmic granules were more diffusely distributed (although still limited to the convoluted tubules) and increased in number within many of the cells. Furthermore, many granules in the dosed males were rectangular rather than round. These differences were detectable at all doses for groups receiving necropsies at either 24 or 72 hours after the last dose. The intracytoplasmic granules in vehicle control and stained positively for alfa 2µ-globulin with the immunohistochemical stain.

Relevance of carcinogenic effects / potential:

It has been demonstrated that renal lesions, which were observed in the NTP study, resulted from the accumulation of aggregates of α 2µ-globulin (a low molecular-weight protein synthesized in the liver) and d-limonene metabolites, probably the 1,2-epoxide, in the P2 segment of the renal proximal tubule (Capen et al., 1999). These aggregates prevent lysosomal degradation, which leads to accumulation in the cytoplasm of the protein or the protein– chemical complex, which leads to single cell necrosis, regenerative tubular proliferation and ultimately, renal neoplasia (Lehman-McKeeman et al., 1990; Hildebrand et al., 1997). This phenomenon has only been observed in the male rat and is not relevant to humans (Strasser et al., 1988; Borghoff et al., 1990; Capen et al., 1999).

References:
Borghoff, S.J., Short, B.G., Swenberg, J.A., 1990. Biochemical mechanisms and pathobiology of nephropathy. Annu. Rev. Pharmacol. Toxicol. 30, 349–367.
Capen, C.C., Dybing, E., Rice, J.M., Wilbourn, J.D., 1999. IARC Consensus: Species differences in thyroid, kidney and urinary bladder carcinogensis. IARC Sci. Publ. 147. International Agency for Research on Cancer, Lyon, France (pp. 175–189).
Hildebrand, H., Hartmann, E., Popp, A., Bomhard, E., 1997. Quantitation of a2- microglobulin after administration of structurally divergent chemical compounds. Arch. Toxicol. 71, 351–359.
Lehman-McKeeman, L., Caudill, D., Takigiku, R., Schneider, R., Young, J.A., 1990. Comparative disposition of D-limonene in rats and mice. relevance to male-ratspecific nephrotoxicity. Toxicol. Lett. 53, 193–195.
Strasser Jr., J., Charbonneau, M., Borghoff, S.J., Turner, M.J., Swenberg, J.A., 1988. Renal protein droplet formation in male Fischer 344 rats after isophorone (IPH) treatment. Toxicologist 8, 136.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Remarks on result:

other: Effect type: toxicity (migrated information)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Remarks on result:

other: Effect type: toxicity (migrated information)

Dose descriptor:

NOAEL

Based on:

test mat.

Sex:

male

Remarks on result:

not determinable

Remarks:

no NOAEL identified. Effect type:carcinogenicity (migrated information)

Dose descriptor:

NOAEL

Effect level:

600

Based on:

test mat.

Sex:

female

Remarks on result:

other: Effect type: carcinogenicity (migrated information)

Conclusions:

Under the conditions of these 2-year gavage studies, there was clear evidence of carcinogenic activity of d-limonene for male F344/N rats, as shown by increased incidences of tubular cell hyperplasia, adenomas, and adenocarcinomas of the kidney. There was no evidence of carcinogenic activity of d-limonene for female F344/N rats that received 300 or 600 mg/kg. The findings in male rats have been accepted not to be relevant for humans.

Executive summary:

A two-year carcinogenicity study was conducted with d-limonene in corn oil in F344/N rats by daily oral gavage dosing at 0, 75, or 150 mg/kg in males and 0, 300, or 600 mg/kg in females. These doses were selected based on compound-related, potentially life-threatening kidney lesions observed in males at 300 mg/kg and higher and on the large number of deaths of female rats at 2400 mg/kg. No compound-related clinical signs were reported for the duration of the study. Mean body weights of rats dosed with d-limonene were similar to those of vehicle controls throughout the studies. Survival of the high-dose male group was significantly greater than that of the vehicle controls after week 81, whereas survival of the high-dose female group was significantly lower than that of the vehicle controls after week 39. In the 2-year studies, the kidney was confirmed as the primary target organ for chemically related lesions. No lesions were observed in female rats. For males, the non-neoplastic lesions included exacerbation of the age-related nephropathy, linear deposits of minerals in the renal medulla and papilla, and focal hyperplasia of the transitional epithelium overlying the renal papilla. Uncommon tubular cell adenomas and adenocarcinomas of the kidney also occurred in dosed male rats, and this effect was supported by a dose-related increased incidence of tubular cell hyperplasia. Further microscopic examination of the kidney sections from these rats indicated a compound-related increase in intracytoplasmic granules in the proximal convoluted tubules of dosed male rats but not of female rats. The granules were shown to contain alfa 2µ-globulin.

Under the conditions of these 2 -year gavage studies, there was clear evidence of carcinogenic activity of d-limonene for male F344/N rats, as shown by increased incidences of tubular cell hyperplasia, adenomas , and adenocarcinomas of the kidney. The findings in male rats have meanwhile been accepted not to be relevant for humans, therefore NOAELs for toxicity and carcinogencity can be established at 150 mg/kg bw in males and 600 mg/kg bw in females.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

150 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

High quality study

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 available data and current knowledge on rat specific carcinogenesis, Depanol I is not considered carcinogenic for humans and does not have to be classified for carcinogenicity according the EU labelling regulations Commission Directive 93/21/EEC or CLP regulation No. 1272/2008 of 16 December 2008.

Additional information

As d-limonene was used for read across of the repeated dose toxicity, carcinogenicity with d-limonene was also reported.

Justification for read across based on the ECHA guidance is provided in Section 13.

A key two-year carcinogenicity study was conducted with d-limonene in corn oil in F344/N rats by daily oral gavage dosing at 0, 75, or 150 mg/kg in males and 0, 300, or 600 mg/kg in females (NTP / Jameson, 1990). No compound-related clinical signs were reported for the duration of the study. Mean body weights of rats dosed with d-limonene were similar to those of vehicle controls throughout the studies. Survival of the high-dose male group was significantly greater than that of the vehicle controls after week 81, whereas survival of the high-dose female group was significantly lower than that of the vehicle controls after week 39. In the 2-year studies, the kidney was confirmed as the primary target organ for chemically related lesions. No lesions were observed in female rats. For males, the non-neoplastic lesions included exacerbation of the age-related nephropathy, linear deposits of minerals in the renal medulla and papilla, and focal hyperplasia of the transitional epithelium overlying the renal papilla. Uncommon tubular cell adenomas and adenocarcinomas of the kidney also occurred in dosed male rats, and this effect was supported by a dose-related increased incidence of tubular cell hyperplasia. Further microscopic examination of the kidney sections from these rats indicated a compound-related increase in intracytoplasmic granules in the proximal convoluted tubules of dosed male rats but not of female rats. The granules were shown to contain alfa 2µ-globulin. Under the conditions of these 2 -year gavage studies, there was clear evidence of carcinogenic activity of d-limonene for male F344/N rats, as shown by increased incidences of tubular cell hyperplasia, adenomas , and adenocarcinomas of the kidney. The findings in male rats have meanwhile been accepted not to be relevant for humans, therefore NOAELs for toxicity and carcinogencity can be established at 150 mg/kg bw in males and 600 mg/kg bw in females.

A supporting two-year carcinogenicity study with d-limonene was also conducted in corn oil in B6C3F1 mice by oral gavage at 0, 250, or 500 mg/kg in males and 0, 500, or 1000 mg/kg in females. No compound-related clinical signs were reported for the duration of the study.

Mean body weights of dosed and vehicle control male mice were similar throughout the studies. Mean body weights of high dose female mice were notably lower than those of the vehicle controls after week 28. No relevant difference in survival was observed between vehicle control and dosed male and female mice.

In mice, no chemically related increases in neoplasms were observed. Under the conditions of these 2 -year gavage studies, there was no evidence of carcinogenic activity of d-limonene for male B6C3F1 mice that received 250 or 500 mg/kg. There was no evidence of carcinogenic activity of d-limonene for female B6C3F1 mice that received 500 or 1000 mg/kg.

Justification for selection of carcinogenicity via oral route endpoint:
D-limonene was used as source chemical. The study in rats was selected, which is considered as main species: the study in mice is considered as supporting species.