(R)-p-mentha-1,8-diene

Carcinogenicity

Description of key information

d‑Limonene has been studied in carcinogenicity studies in F344/N rats and B6C3F1 mice ( 2-year study by gavage).

Increased incidences of renal tubular cell hyperplasia, adenomas, and adenocarcinomas of the kidney have been observed in male rats.

No increased incidences of tumors have been reported in female rats and in mice.
The mechnaism of nephrocarcinogenicity has been proven as being male-rat specific and not relevant for humans.

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:

From February 16, 1981 to February 17, 1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

GLP study performed similarly to OECD Guideline 451 but with deviations: dosing 5 days/week instead of 7 days/week; food consumption and clinical biochemical tests not followed

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Deviations:

yes

Remarks:

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

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

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, USA)
- Age at study initiation: 8-9 weeks
- Weight at study initiation: Males: 30.2-30.3 g; females: 21.2-22.0 g
- Housing: Housed in groups of five in polycarbonate cages
- Diet (e.g. ad libitum): NIH 07 Rat and Mouse Ration (Zeigler Bros., Inc., Gardners, PA, USA), ad libitum
- Water (e.g. ad libitum): Automatic watering system (Edstrom Industries, Waterford, USA), ad libitum
- Acclimation period: 19 days

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: Appropriate amount of test substance was weighed and mixed with corn oil by shaking in a volumetric flask.

VEHICLE
- Amount of vehicle (if gavage): 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Apparatus: Periodic analysis for d-limonene in dose preparations 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/min, and a flame ionization detector.
- Sampling frequency: After every 8 weeks
- Results: 87-110% of the target concentrations

Duration of treatment / exposure:

103 weeks

Frequency of treatment:

Once per day; 5 days/week

Post exposure period:

One week

Remarks:

Doses / Concentrations:
Male: 0, 250 and 500 mg/kg bw/day; female: 0, 500 and 1000 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: Doses were selected based on the deaths observed for both male and female mice at 2000 mg/kg bw/day during the 13-week studies and the body weight depression in male mice at 1000 mg/kg bw/day and higher.
- Rationale for animal assignment (if not random): Random

Positive control:

No

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: Once per week for 12 weeks and once per month thereafter

Sacrifice and pathology:

GROSS PATHOLOGY: Yes; necropsy performed on all animals
HISTOPATHOLOGY: Yes; performed on all animals; histologic exams performed on all animals dying during the studies, all vehicle controls and all high dose animals. Tissues examined include: adrenal glands, brain, cecum, colon, costochondral junction, duodenum, epididymis/seminal vesicles/tunica vaginalis/scrotal sac/prostate/testes or ovaries/uterus, esophagus, eyes, femur or sternebrae or vertebrae including marrow, gallbladder, gross lesions and tissue masses with regional lymph nodes, heart, ileum, jejunum, kidneys, larynx and pharynx, liver, lungs and bronchi, mammary gland, mandibular and mesenteric lymph nodes, nasal cavity and turbinates, oral cavity, pancreas, parathyroids, pituitary gland, preputial or clitoral gland, rectum, salivary glands, sciatic nerve, skin, spinal cord, spleen, stomach, thigh muscle, thymus, thyroid gland, trachea, urinary bladder and Zymbal gland. Tissues examined in low dose groups include adrenal glands, kidney, liver and spleen for female mice.

Other examinations:

None

Statistics:

- Survival: 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.
- Neoplasm and nonneoplastic lesion incidences: Incidental tumor analysis, life table test (Cox, 1972; Tarone, 1975), Fisher exact test and the Cochran-Armitage trend test (Armitage, 1971; Gart et al., 1979) were used to assess neoplasm and nonneoplastic lesion prevalence.

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 specified

Haematological findings:

not specified

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:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

MORTALITY:
- Survival of the low dose group of male mice was significantly lower than that of the vehicle controls at the end of the study.
- Survival in males at week 104: 33/50, 24/50 and 39/50 animals at 0, 250 and 500 mg/kg bw/day, respectively.
- Survival in females at week 104: 43/50, 44/50 and 43/50 animals at 0, 500 and 1000 mg/kg bw/day, respectively.
- See table 1 for more data

CLINICAL SIGNS:
- No treatment-related clinical signs were observed during the study.

BODY WEIGHT AND WEIGHT GAIN
- Mean bodyweights of high dose female mice were 5-15% lower than those of the vehicle controls after week 28.
- Mean bodyweights of dosed and vehicle control male mice were similar throughout the studies.

HISTOPATHOLOGY: NON-NEOPLASTIC/ NEOPLASTIC:
- No treatment-related statistically or biologically significant effects were observed during the study.

Relevance of carcinogenic effects / potential:

Not relevant as no evidence of carcinogenic activity of d-limonene for male and female B6C3F1 mice.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 250 - <= 500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no evidence of carcinogenic activity

Remarks on result:

not determinable

Remarks:

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

Key result

Dose descriptor:

NOAEL

Effect level:

>= 500 - <= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no evidence of carcinogenic activity

Remarks on result:

not determinable

Remarks:

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

Table 1: Survival of mice in the 2-year gavage studies of d-limonene

 

	Vehicle Control	250 mg/kg bw/day	500 mg/kg bw/day	1000 mg/kg bw/day
MALE (a)
Animals initially in study	50	50	50
Nonaccidental deaths before termination (b)	14	24	9
Accidentally killed	2	2	2
Animals missing	1	0	0
Killed at termination	33	24	38
Died during termination period	0	0	1
Survival P values (c)	0.361	0.048	0.348
FEMALE (a)
Animals initially in study	50		50	50
Nonaccidental deaths before termination (b)	7		5	7
Accidentally killed	0		1	0
Killed at termination	42		44	42
Died during termination period	1		0	1
Survival P values (c)	1		0.757	0.995

(a) Termination period: week 104

(b) Includes animals killed in a moribund condition

(c) The result of the life table trend test is in the vehicle control column, and the results of the life table pairwise comparisons with the vehicle controls are in the dosed columns.

Conclusions:

There was no evidence of carcinogenic activity of d-limonene for male and female B6C3F1 mice.

Executive summary:

In a 2-year carcinogenicity study performed similarly to OECD Guideline 451 and in compliance with GLP, d-limonene was administered through gavage to groups of 50 B6C3F1 mice/sex/dose mixed in corn oil at dose levels of 0, 250 and 500 mg/kg bw/day (in males) or 0, 500 and 1000 mg/kg bw/day (in females) for 103 weeks (5 days/week). Animals were observed twice daily for clinical signs of toxicity and bodyweights were recorded once per week for 12 weeks and once per month thereafter. Necropsy performed on all animals and microscopic examination of specified tissues was performed for all animals dying during the studies, all vehicle controls and all high dose animals at the end of the treatment period.

 

Survival of the low dose group of male mice was significantly lower than that of the vehicle controls at the end of the study. Survival at week 104 was 33/50 male and 43/50 female in vehicle control group; 24/50 males and 44/50 females in low dose group and 39/50 males and 43/50 females in high dose group. No treatment-related clinical signs were observed during the study. Mean bodyweights of high dose female mice were 5-15% lower than those of the vehicle controls after week 28. Mean bodyweights of dosed and vehicle control male mice were similar throughout the studies. No treatment-related statistically or biologically significant histologic effects were observed during the study.

 

Therefore, there was no evidence of carcinogenic activity of d-limonene for male and female B6C3F1 mice.

Reference 2

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From February 02, 1981 to February 11, 1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

GLP study performed similarly to OECD Guideline 451 but with deviations: dosing 5 days/week instead of 7 days/week; food consumption and clinical biochemical tests not followed

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Deviations:

yes

Remarks:

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

Principles of method if other than guideline:

Not applicable

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, USA)
- Age at study initiation: 7-8 weeks
- Weight at study initiation: Males: 183-187 g; females: 132-133 g
- Housing: Housed in groups of five in polycarbonate cages
- Diet (e.g. ad libitum): NIH 07 Rat and Mouse Ration (Zeigler Bros., Inc., Gardners, PA, USA), ad libitum
- Water (e.g. ad libitum): Automatic watering system (Edstrom Industries, Waterford, USA), ad libitum
- Acclimation period: 19 days

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: Appropriate amount of test substance was weighed and mixed with corn oil by shaking in a volumetric flask.

VEHICLE
- Amount of vehicle (if gavage): 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Apparatus: Periodic analysis for d-limonene in dose preparations 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/min, and a flame ionization detector.
- Sampling frequency: After every 8 weeks
- Results: 87-110% of the target concentrations

Duration of treatment / exposure:

103 weeks

Frequency of treatment:

Once per day; 5 days/week

Post exposure period:

1 week in males; 1-2 weeks in females

Remarks:

Doses / Concentrations:
Male: 0, 75 and 150 mg/kg bw/day; female: 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: Doses were selected based on compound-related, potentially life-threatening kidney lesions observed in males at 300 mg/kg bw/day and higher and on the large number of deaths of female rats at 2400 mg/kg bw/day during the 13-week study.
- Rationale for animal assignment (if not random): Random

Positive control:

No

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: Once per week for 12 weeks and once per month thereafter

Sacrifice and pathology:

GROSS PATHOLOGY: Yes; necropsy performed on all animals
HISTOPATHOLOGY: Yes; performed on all animals; histologic exams performed on all animals dying during the studies, all vehicle controls, all low dose female rats and all high dose animals. Tissues examined include: adrenal glands, brain, cecum, colon, costochondral junction, duodenum, epididymis/seminal vesicles/tunica vaginalis/scrotal sac/prostate/testes or ovaries/uterus, esophagus, eyes, femur or sternebrae or vertebrae including marrow, gross lesions and tissue masses with regional lymph nodes, heart, ileum, jejunum, kidneys, larynx and pharynx, liver, lungs and bronchi, mammary gland, mandibular and mesenteric lymph nodes, nasal cavity and turbinates, oral cavity, pancreas, parathyroids, pituitary gland, preputial or clitoral gland, rectum, salivary glands, sciatic nerve, skin, spinal cord, spleen, stomach, thigh muscle, thymus, thyroid gland, trachea, urinary bladder and Zymbal gland. Tissues examined in low dose groups include adrenal glands, kidney, liver, spleen and testis for male rats.

Other examinations:

None

Statistics:

- Survival: 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.
- Neoplasm and nonneoplastic lesion incidences: Incidental tumor analysis, life table test (Cox, 1972; Tarone, 1975), Fisher exact test and the Cochran-Armitage trend test (Armitage, 1971; Gart et al., 1979) were used to assess neoplasm and nonneoplastic lesion prevalence.

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:

no effects observed

Haematological findings:

no effects observed

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:

MORTALITY:
- Survival of the high dose female rats after week 39 and of the vehicle control male rats after week 81 was significantly reduced.
- Survival in males at week 104: 29/50, 33/50 and 40/50 animals at 0, 75 and 150 mg/kg bw/day, respectively.
- Survival in females at week 104: 42/50, 40/50 and 26/50 animals at 0, 300 and 600 mg/kg bw/day, respectively.
- Several animals died accidentally.

CLINICAL SIGNS:
- No treatment-related clinical signs were observed during the study.

BODY WEIGHT AND WEIGHT GAIN
- Mean body weights of high dose rats were generally 4-7% lower than those of the vehicle controls from week 2 (in males) or 28 (in females) to the end of the studies.

HISTOPATHOLOGY:

Kidney:
- Non-neoplastic effects: No lesions were observed in female rats. For males, the nonneoplastic lesions included exacerbation of the age-related nephropathy, linear deposits of mineral in the renal medulla and papilla (in 7/50, 43/50 and 48/50 rats at 0, 75 and 150 mg/kg bw/day, respectively), and focal hyperplasia of the transitional epithelium overlying the renal papilla (in 0/50, 35/50 and 43/50 rats at 0, 75 and 150 mg/kg bw/day, respectively).
- Neoplastic effects: Uncommon tubular cell adenomas (in 0/50, 4/50 and 8/50 rats at 0, 75 and 150 mg/kg bw/day, respectively) and adenocarcinomas (in 0/50, 4/50 and 3/50 rats at 0, 75 and 150 mg/kg bw/day, respectively) of the kidney occurred in dosed male rats, and this effect was supported by a dose-related increased incidence of tubular cell hyperplasia (in 0/50, 4/50 and 7/50 rats at 0, 75 and 150 mg/kg bw/day, respectively)

Uterus, testis, hematopoietic system, skin, subcutaneous tissue and eye:
- No treatment-related statistically or biologically significant effects were observed during the study.

Relevance of carcinogenic effects / potential:

This mechanism of nephrocarcinogenicity has been proven as being nale-rat specific and not relevant for humans.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 75 - <= 150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: clear evidence of carcinogenic activity based on the increased incidences of tubular cell hyperplasia, adenomas and adenocarcinomas of the kidney

Remarks on result:

not determinable

Remarks:

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

Key result

Dose descriptor:

NOAEL

Effect level:

>= 300 - <= 600 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no evidence of carcinogenic activity

Remarks on result:

not determinable

Remarks:

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

Table 1: Survival of rats in the 2-year gavage studies of d-limonene

 

	Vehicle Control	Low Dose	High Dose
MALE (a)		75 mg/kg bw/day	150 mg/kg bw/day
Animals initially in study	50	50	50
Nonaccidental deaths before termination (b)	20	16	5
Accidentally killed	1	1	5
Killed at termination	29	33	40
Survival P values (c)	0.001	0.497	0.001
FEMALE (a)		300 mg/kg bw/day	600 mg/kg bw/day
Animals initially in study	50	50	50
Nonaccidental deaths before termination (b)	5	8	16
Accidentally killed	3	2	8
Killed at termination	42	39	24
Died during termination period	0	1	2
Survival P values (c)	0.003	0.571	0.006

(a) Termination period: male--week 104; female--weeks 104-105

(b) Includes animals killed in a moribund condition

(c) The result of the life table trend test is in the vehicle control column, and the results of the life table pairwise comparisons with the vehicle controls are in the dosed columns.

 

Table 2: Incidences of male rats with renal lesions in the 2-year gavage study of d-limonene

 

Site/Lesion	Vehicle Control	75mg/kg bw/day	150 mg/kg bw/day
Renal papilla
Mineralization	7/50	43/50	48/50
Epithelial hyperplasia	0/50	35/50	43/50
Kidney
Tubular cell hyperplasia	0/50	4/50	7/50
Tubular cell adenoma	0/50	4/50	8/50
Tubular cell adenocarcinoma	0/50	4/50	3/50

Conclusions:

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.

Executive summary:

In a 2 year carcinogenicity study performed similarly to OECD Guideline 451 and in compliance with GLP, d-limonene was administered through gavage to groups of 50 F344/N rats/sex/dose mixed in corn oil at dose levels of 0, 75 and 150 mg/kg bw/day (in males) or 0, 300 and 600 mg/kg bw/day (in females) for 103 weeks (5 days/week). Animals were observed twice daily for clinical signs of toxicity and bodyweights were recorded once per week for 12 weeks and once per month thereafter. Necropsy performed on all animals and microscopic examination of specified tissues was performed for all animals dying during the studies, all vehicle controls, all low dose female rats and all high dose animals at the end of the treatment period.

 

Survival of the high dose female rats after week 39 and of the vehicle control male rats after week 81 was significantly reduced. Survival at week 104 was 29/50 male and 42/50 female in vehicle control group; 33/50 males and 40/50 females in low dose group and 40/50 males and 20/50 females in high dose group. Mean body weights of high dose rats were generally 4-7% lower than those of the vehicle controls from week 2 (in males) or 28 (in females) to the end of the studies. No treatment-related clinical signs were observed during the study. Kidney was confirmed as the primary target organ for chemically related lesions. No lesions were observed in female rats. For males, the nonneoplastic lesions included exacerbation of the age-related nephropathy, linear deposits of mineral 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.

 

There was 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. However, this mechanism of nephrocarcinogenicity has been proven as being male-rat specific and not relevant for humans.

Reference 3

Endpoint:

carcinogenicity

Remarks:

other: oral (feed) or dermal

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study performed to assess the skin promotion tumour activity odf substance, which is scientifically acceptable. No data on mortality and clinical observations; study performed with 24 females/dose instead of 50 mice/sex/dose; actual doses (mg/kg bw/day) not reported; individual animal data not reported

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Principles of method if other than guideline:

40-week carcinogenicity study: two-stage skin carcinogenesis model

GLP compliance:

not specified

Species:

mouse

Strain:

CD-1

Sex:

female

Route of administration:

other: oral (feed) or dermal

Vehicle:

acetone

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

40 weeks

Frequency of treatment:

- Dietary test: Daily, ad libitum
- Topical test: Twice per week

Post exposure period:

No data

Remarks:

Doses / Concentrations:
1% w/w
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
0.2 mL d-limonene (630 nmol, 1:1) in acetone
Basis:
other: applied to shaved skin

No. of animals per sex per dose:

24 females

Control animals:

yes, concurrent vehicle

Dose descriptor:

NOAEL

Sex:

female

Basis for effect level:

other: no clear evidence of skin tumor promoting activity

Remarks on result:

not determinable

Remarks:

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

Skin papillomas:

- AlI mice applied once with DMBA and subsequently applied twice weekly with TPA rapidly developed papillomas.

- Only few mice applied once with DMBA and subsequently applied twice weekly with d-limonene developed papillomas and significant promotion by d-limonene could not be detected until week 34 post-initiation.

- No papillomas developed in other treatment groups.

 

Bodyweight gain and diet consumption:

- No significant treatment-related effects were observed during the study.

Conclusions:

There was no clear evidence of skin tumor promoting activity of d-limonene for female CD-1 mice.

Executive summary:

In a 40-week carcinogenicity study, d-limonene was administered either topically or via the diet to groups of female CD-1 mice (24/dose) in a two-stage skin carcinogenesis model. Tumors were initiated (Day 0) by the application of 0.2 µmol (51.2 µg) DMBA (7,12-dimethylbenz[a]anthracene) in 0.2 mL of acetone to the shaved backs of the mice. Control mice were treated with 0.2 mL acetone. In the dietary test, the test material was incorporated (1% by weight) into the basal diet on Day 7. Beginning on Day 14 and twice a week thereafter for the duration of the study the topically-tested mice were treated by the application to the shaved area of one of the following: 0.2 mL acetone; 10 nmol TPA (12-O-tetradecanoylphorbol-13-acetate) in 0.2 mL acetone or 0.2 mL d-limonene (630 nmol, 1: 1) in acetone. Beginning the seventh week following DMBA application, the number of mice bearing papillomas and the number of papillomas per treatment group were recorded at weekly intervals. Carcinomas were recorded grossly us downward-invading lesions. Carcinoma-bearing mice were killed. The lesions were fixed, embedded, sectioned and examined histologically and their malignancy confirmed. Mice were weighed at weekly intervals prior to week 10 post-initiation; thereafter they were weighed at 2-week intervals. Diet intake was monitored during the initial 4 weeks of the study.

 

All mice applied once with DMBA and subsequently applied twice weekly with TPA rapidly developed papillomas. Only few mice applied once with DMBA and subsequently applied twice weekly with d-limonene developed papillomas and significant promotion by d-limonene could not be detected until week 34 post-initiation. No papillomas developed in other treatment groups. No significant treatment-related effects were observed on bodyweight gain and diet consumption during the study.

 

Therefore, there was no clear evidence of skin tumor promoting activity of d-limonene for female CD-1 mice.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

75 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

CLP Annex I, 3.9.2.8.1. (e) states that d-limonene induces a male-rat specific carcinogenic effect on kidneys, which is not deemed relevant for classification of d-limonene as a carcinogenic substance.

Additional information

d‑Limonene has been studied in carcinogenicity studies in F344/N rats and B6C3F1 mice (2-year study by gavage). Increased incidences of renal tubular cell hyperplasia, adenomas, and adenocarcinomas of the kidney have been observed in male rats. No increased incidences of tumors have been reported in female rats and in mice. The mechanism of nephrocarcinogenicity has been proven as being male-rat specific and not relevant for humans.

Mechanisms and specificity of toxicity of d-limonene on kidney of male rats and its non relevance for human are well known.

d-limonene produces hyaline droplets, tubular necrosis, regeneration, hyperplasia adenomas and carcinomas in the kidneys of male rats. These effects are not observed in mice. The key events are:

- Metabolism to d-limonene epoxide,

- Binding of the metabolite to serum alpha2u-globulin, which appears to be the rate-limiting step in the development of the nephropathy and tumours,

- Accumulation of alpha2u-globulin in the form of hyaline droplets in the renal tubules,

- Chronic renal cellular protein overload,

- Renal cell necrosis and compensatory cell proliferation, which is the critical event and must be sustained for tumours to develop,

- Renal tubule adenomas and carcinomas.

The key events can be demonstrated by measurement of d-limonene epoxide in plasma, demonstration of alpha2u-globulin in renal tubules and histology of kidney tumours. Alpha2u-globulin is a male rat specific protein with no equivalent in mice or humans capable of binding d-limonene epoxide.Thus, the conclusion of the assessment is that the proven mechanism of action is not relevant for humans. (Crome S. et al., 2008, Developments in Lifesciences Vol B No.2)

A study was also available, which showed that there was no clear evidence of skin tumour promoting activity of d-limonene for female CD-1 mice.