Orange, sweet, ext.

Carcinogenicity

Description of key information

Carcinogenic bioassay (RA from d-limonene, equivalent or similar to OECD 451):
No evidence of carcinogenic activity in mice and rats

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:

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. Due to the read-across purpose it was given a Klimisch 2 rating, in accordance with the ECHA Practical guide #6 on the reporting of read-across in IUCLID. The justification for read across is provided in the attached background material of the chapter summary.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same 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

Dose / conc.:

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

Remarks:

male/female

Dose / conc.:

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

Remarks:

Male

Dose / conc.:

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

Remarks:

Male/Female

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Female

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

Description (incidence and severity):

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

Mortality:

mortality observed, treatment-related

Description (incidence):

- 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

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- 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.

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

Description (incidence and severity):

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

Histopathological findings: neoplastic:

no effects observed

Relevance of carcinogenic effects / potential:

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

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)

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:

Under the test conditions, 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.

 

Under the test conditions, 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:

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. Due to the read-across purpose it was given a Klimisch 2 rating, in accordance with the ECHA Practical guide #6 on the reporting of read-across in IUCLID. The justification for read across is provided in the attached background material of the chapter summary.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same 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

Dose / conc.:

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

Remarks:

Male/Female

Dose / conc.:

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

Remarks:

Male

Dose / conc.:

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

Remarks:

Male

Dose / conc.:

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

Remarks:

Female

Dose / conc.:

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

Remarks:

Female

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

Description (incidence and severity):

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

Mortality:

mortality observed, treatment-related

Description (incidence):

- 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.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- 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.

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

Description (incidence and severity):

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).

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

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Kidney:
- 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)

Relevance of carcinogenic effects / potential:

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

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)

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:

Under the test conditions, 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.

 

Under the test conditions, 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. This mechanism of nephrocarcinogenicity has been proven as being nale-rat specific and not relevant for humans.

Reference 3

Endpoint:

carcinogenicity: oral

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

See attached justification

Reason / purpose for cross-reference:

read-across source

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

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

Mortality:

mortality observed, treatment-related

Description (incidence):

- 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

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- 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.

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

Description (incidence and severity):

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

Histopathological findings: neoplastic:

no effects observed

Relevance of carcinogenic effects / potential:

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

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)

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:

Under the test conditions, 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.

 

Under the test conditions, there was no evidence of carcinogenic activity of d-limonene for male and female B6C3F1 mice.

Reference 4

Endpoint:

carcinogenicity: oral

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

See attached justification

Reason / purpose for cross-reference:

read-across source

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

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

Mortality:

mortality observed, treatment-related

Description (incidence):

- 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.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- 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.

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

Description (incidence and severity):

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).

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

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Kidney:
- 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)

Relevance of carcinogenic effects / potential:

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

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)

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:

Under the test conditions, 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.

 

Under the test conditions, 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. This mechanism of nephrocarcinogenicity has been proven as being nale-rat specific and not relevant for humans.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

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 results of the key studies, orange oil does not need to be classified for carcinogenicity when considering the criteria outlined in Annex I of 1272/2008/EC .

Additional information

One carcinogenicity study (equivalent or similar to OECD 451, read-across from d-limonene to orange oil) considering two species was available. Both mice and rats were tested.

In the experiment with B6C3F1 mice there was found no evidence of carcinogenic activity of d-limonene for both males and females. The experiment with F344/N rats found clear evidence of carcinogenic activity of d-limonene for males, as shown by increased incidences of tubular cell hyperplasia, adenomas, and adenocarcinomas of the kidney. However, the observed effect was considered to be male rat specific (nephrocarcinogenicity) and therefore not relevant for human. There was no evidence of carcinogenic activity of d-limonene for female F344/N rats. In conclusion, both experiment indicate that there is no evidence for carcinogenicity of orange oil.