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

Carcinogenicity

Description of key information

No studies were located on l-limonene. 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:

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: Read-across from an analogue substance for which there is available information (Klimish =2).

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
See cross-reference to justification of read-across.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

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

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

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

Conclusions:

Based on the read-across from the analogue substance d-limonene, l-limonene is not expected to have any carcinogenic activity in male and female B6C3F1 mice.

Executive summary:

A 2 -year carcinogenicity study was performed similarly to OECD Guideline 451 and in compliance with GLP with the analogue substance d-limonene which 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).  

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.

Based on the read-across from the analogue substance d-limonene, l-limonene is not expected to have any carcinogenic activity in male and female B6C3F1 mice.

Reference 2

Endpoint:

carcinogenicity: oral

Type of 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: Read-across from an analogue substance for which there is available information (Klimish=4).

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
See cross-reference to justification of read-across.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

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

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

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

75 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

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. This effect is well known to be specifics of male rats and irrelevant to humans.

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 male-rat specific and not relevant for humans.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

75 mg/kg bw/day

Study duration:

chronic

Species:

rat

System:

urinary

Organ:

kidney

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

No studies were located on l-limonene.For further information on read-across justification, see section 13: point "read-across approach"

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.

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.

Justification for classification or non-classification

Harmonized classification:

No harmonized classification is available according to the Regulation (EC) No 1272/2008.

Self classification:

CLP Annex I, 3.9.2.8.1. (e) states that d-limonene induces a male-rat specific carcinogenic effect on kidney, which is not deemed relevant for classification of d-limonene as a carcinogenic substance. Therefore, l-limonene does not need to be classified for carcinogenicity.