Registration Dossier

Administrative data

Description of key information

There was no data on l-limonene repeated dose toxicity. Therefore, the read-across from the analogue substance d-limonene is applied.

When administered orally by gavage for at least 6 months, d-limonene induces some toxicological effects from 1000 mg/kg bw/d. At this dose level, following 90 days of exposure, d-limonene induces decreased bodyweight gain and clinical signs in mice. 180 days of exposure to d-limonene at this dose level decreased bodyweight gain and increased relative and absolute kidney weights of dogs (with protein casts in the renal tubules of females).

The most relevant LOAEL value to derive the DNEL is considered to be 1000 mg/kg bw from the 180-d dog study.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: 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 data (Klimish =2).
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
See cross-reference to justification of read-across.
Reason / purpose:
read-across source
Reason / purpose:
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 examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
825 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
mortality
body weight and weight gain
Remarks on result:
other: Based on the read-across from the analogue substance d-limonene.
Key result
Dose descriptor:
LOAEL
Effect level:
1 650 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
Remarks on result:
other: Based on the read-across from the analogue substance d-limonene.
Key result
Dose descriptor:
NOAEL
Effect level:
1 650 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
mortality
body weight and weight gain
Remarks on result:
other: Based on the read-across from the analogue substance d-limonene.
Key result
Dose descriptor:
LOAEL
Effect level:
3 300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: Based on the read-across from the analogue substance d-limonene.
Key result
Critical effects observed:
not specified
Conclusions:
Based on the read-across from the analogue substance d-limonene, the NOAEL of l-limonene for male and female rats was estimated to be 825 and 1650mg/kg-bw/day and the LOAEL for male and female rats was considered to be 1650 and 3300 mg/kg-bw/day based on decreased bodyweight gains.
Executive summary:

A 16-day subacute toxicity study was conducted similarly to OECD Guideline 407 and in compliance with GLP with the analogue substance d-limonene which was was administered through gavage to groups of 5 F344/N rats/sex/dose mixed in corn oil at dose levels of 0, 413, 825, 1650, 3300 and 6600 mg/kg bw/day for 16 days (5 days/week).

All rats that received 6600 mg/kg bw/day and 5/5 males and 3/5 females that received 3300 mg/kg bw/day d-limonene died within the first 2 days. The final mean body weight of male rats that received 1650 mg/kg bw/day was 10% lower than that of the vehicle controls. The final mean body weight of female rats that received 3300 mg/kg bw/day was 8% lower than that of the vehicle controls. No treatment-related clinical signs were observed in rats that received doses of 1650 mg/kg bw/day or lower.No treatment-related lesions were observed.

 

Based on the read-across from the analogue substance d-limonene, the NOAEL of l-limonene for male and female rats was estimated to be 825 and 1650mg/kg-bw/day and the LOAEL for male and female rats was considered to be 1650 and 3300 mg/kg-bw/day based on decreased bodyweight gains.

Endpoint:
sub-chronic toxicity: 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 data (Klimish =2).
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
See cross-reference to justification of read-across.
Reason / purpose:
read-across source
Reason / purpose:
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 examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: mortality at 2000 mg/kg bw/day; decreased bodyweight gain and occurence of clinical signs of toxicity (rough hair coats and decreased activity) at 1000 and 2000 mg/kg bw/day
Key result
Dose descriptor:
LOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
Key result
Critical effects observed:
not specified
Conclusions:
Based on the read-across from the analogue substance d-limonene, the NOAEL of l-limonene was estimated to be 500 mg/kg bw/day. The LOAEL was estimated to be 1000 mg/kg bw/day for both female and male mice, based on observation of clinical signs in both sexes and decreased bodyweights in males.
Executive summary:

A 13-week subchronic toxicity study was performed similarly to OECD Guideline 408 and in compliance with GLP with the analogue substance d-limonene which was administered through gavage to groups of 10 B6C3F1 mice/sex/dose mixed in corn oil at dose levels of 0, 125, 250, 500, 1000 or 2000 mg/kg bw/day for 13 weeks (5 days/week). Animals were observed twice daily for clinical signs of toxicity and bodyweights were recorded weekly. Necropsy performed on all animals and microscopic examination of specified tissues was performed for all control and high dose animals scheduled to be killed at the end of the treatment period.

One of 10 males and 2/10 females that received 2000 mg/kg bw/day and 1/10 females that received 500 mg/kg bw/day died before the end of the studies. Several animals in other groups died as a result of gavage error. Clinical signs of rough hair coats and decreased activity were observed at the two highest doses. Final mean bodyweights of mice that received 1000 or 2000 mg/kg bw/day were 10% lower than that of the vehicle controls for males and 2% lower for females. An alveolar cell adenoma was observed in the lung of 1/10 females that received 2000 mg/kg bw/day.

 

Based on the read-across from the analogue substance d-limonene, the NOAEL of l-limonene was estimated to be 500 mg/kg bw/day. The LOAEL was estimated to be 1000 mg/kg bw/day for both female and male mice, based on observation of clinical signs in both sexes and decreased bodyweights in males.

Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Read-across from an analogue substance for which there is available data (Klimish =2).
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
See cross-reference to justification of read-across.
Reason / purpose:
read-across source
Reason / purpose:
read-across: supporting information
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: increased absolute and relative female kidney weight and relative male kidney weight at 1000 mg/kg bw/day
Remarks on result:
other: Based on the read-across from the analogue substance d-limonene.
Key result
Dose descriptor:
LOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: Based on the read-across from the analogue substance d-limonene.
Key result
Critical effects observed:
not specified
Conclusions:
Based on the read-across from te analogue substance d-limonene, the NOAEL and LOAEL of l-limonene for beagle dogs were estimatedto be 100 and 1000 mg/kg bw/day, respectively, based on the increased absolute and relative female kidney weight and relative male kidney weight.
Executive summary:

A 6-month subchronic toxicity study was performed similarly to OECD Guideline 409 with the analogue substance d-limonene which was administered through gavage to groups of adult beagle dogs (5/sex/dose) at dose levels of 0 (tap water), 0.12 or 1.2 mL/kg bw/day (0, 100 or 1000 mg/kg bw/day) in two divided doses for 180 days. 

Feed consumption and body weight were unaffected by treatment. Clinical signs of toxicity noted were excretion of soft faeces, dose-related occasional mild discomfort during defaecation, sporadic episodes of emesis and diarrhoea. No treatment-related differences other than an increase in serum cholesterol (35%) and serum alkaline phosphatase levels (two-fold increase) at 1000 mg/kg bw/day were observed in male and female dogs. Linear regression analyses indicated a positive dose-related trend for absolute and relative female kidney weight and relative male kidney weight. There were no histopathological changes in the kidneys, evaluated by both haematoxylin and eosin and Mallory-Heidenhain staining that could be associated with the organ-weight changes. Furthermore, there was no evidence of hyaline droplet accumulation or of any other sign of hydrocarbon-induced nephropathy typical of those seen in male rats treated with d-limonene.

 

Based on the read-across from te analogue substance d-limonene, the NOAEL and LOAEL of l-limonene for beagle dogs were estimatedto be 100 and 1000 mg/kg bw/day, respectively, based on the increased absolute and relative female kidney weight and relative male kidney weight.

Endpoint:
short-term repeated dose toxicity: 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 data (Klimish =2).
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
See cross-reference to justification of read-across.
Reason / purpose:
read-across source
Reason / purpose:
read-across: supporting information
No. of animals per sex per dose:
Five
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
1 650 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
Remarks on result:
other: Based on the read-across from the analogue substance d-limonene.
Key result
Dose descriptor:
LOAEL
Effect level:
3 300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
Remarks on result:
other: Based on the read-across from the analogue substance d-limonene.
Key result
Critical effects observed:
not specified
Conclusions:
Based on the read-across from the analogue substance d-limonene, the NOAEL of l-limonene for male and female mice was estimated to be 1650mg/kg-bw/day and the LOAEL was considered to be 3300 mg/kg-bw/day based on increased mortality rates.
Executive summary:

A 16-day subacute toxicity study was performed similarly to OECD Guideline 407 and in compliance with GLP with the analogue substance d-limonene which was administered through gavage to groups of five B6C3F1 mice/sex/dose mixed in corn oil at dose levels of 0, 413, 825, 1650, 3300 and 6600 mg/kg bw/day for 16 days (5 days/week). Animals were observed twice daily for clinical signs of toxicity and bodyweights were recorded weekly. Necropsy performed on all animals and histological examinations were performed on six mice from survivors of highest dose groups.

 

All but one of 20 mice that received 3300 or 6600 mg/kg bw/day died within 3 days. Vehicle control mice gained little or no weight. Slight and not treatment- related bodyweight loss was observed in all treated groups. No compound-related clinical signs were observed in mice that received 1650 mg/kg bw/day and lived to the end of the studies. No treatment-related histopathologic lesions were observed.

 

Based on the read-across from the analogue substance d-limonene, the NOAEL of l-limonene for male and female mice was estimated to be 1650mg/kg-bw/day and the LOAEL was considered to be 3300 mg/kg-bw/day based on increased mortality rates.

Endpoint:
sub-chronic toxicity: 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 data (Klimish =2).
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
See cross-reference to justification of read-across.
Reason / purpose:
read-across source
Reason / purpose:
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 examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Key result
Dose descriptor:
NOAEL
Based on:
test mat.
Sex:
male
Basis for effect level:
other: male-rat specific nephrotoxicity at all dose levels (considered as not relevant for humans)
Remarks on result:
not determinable
Remarks:
no NOAEL identified
Key result
Dose descriptor:
NOAEL
Effect level:
600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: decrease of bodyweight gains at 1200 and 2400 mg/kg bw/day;
Key result
Dose descriptor:
LOAEL
Effect level:
1 200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Effect level:
600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: mortality at 2400 mg/kg bw/day; occurrence of clinical signs of toxicity (rough hair coats, lethargy and excessive lacrimation) at 1200 and 2400 mg/kg bw/day
Key result
Dose descriptor:
LOAEL
Effect level:
1 200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical signs
Key result
Critical effects observed:
not specified
Conclusions:
Based on the read-across from the analogue substance d-limonene, the NOAEL of l-limonene for male and female rats was estimated to be 600 mg/kg bw/day. The LOAEL for female and male rats were considered to be 1200 and 150 mg/kg bw/day, based on observation of clinical signs and nephropathy, respectively. As nephrotoxicity and accumulation of hyaline droplets were observed in male rats at all dose levels, no NOAEL for male rats could be identified in this study.
Executive summary:

A 13-week subchronic toxicity study was performed similarly to OECD Guideline 408 and in compliance with GLP with the analogue substance d-limonene which was administered through gavage to groups of 10 F344/N rats/sex/dose mixed in corn oil at dose levels of 0, 150, 300, 600, 1200 and 2400 mg/kg bw/day for 13 weeks (5 days/week).  

Five of 10 males and 9/10 female rats that received 2400 mg/kg bw/day died during week 1. Final mean body weights of male rats in 600, 1200 or 2400 mg/kg bw/day groups were 6, 12 or 23% lower than that of the vehicle controls. Final body weight of the female rat that received 2400 mg/kg bw/day and lived to end of the study was 11% lower than the mean of the vehicle controls. Rough hair coats, lethargy and excessive lacrimation were observed at 1200 or 2400 mg/kg bw/day. No treatment-related histopathologic lesions were observed in female rats. Nephropathy was identified in all groups of male rats, and there was a dose-related increased severity of the lesion in dosed groups. Nephropathy was characterized by degeneration of epithelium in the convoluted tubules, granular casts within tubular lumens, primarily in the outer stripe of the outer medulla, and regeneration of the tubular epithelium. Hyaline droplets (protein reabsorption droplets) were observed in the epithelium of proximal convoluted tubules in all groups of male rats, including vehicle controls. This mechanism of nephrocarcinogenicity has been proven as being male-rat specific and not relevant for humans.

Based on the read-across from the analogue substance d-limonene, the NOAEL of l-limonene for female rats was considered to be 600 mg/kg bw/day. When considering the non relevance of the nephrotoxic effects for humans, the NOAEL for male rats would be 600 mg/kg bw/day, based on decrease of bodyweight gains at 1200 and 2400 mg/kg bw/day. The LOAEL for female and male rats were considered to be 1200 and 150 mg/kg bw/day, based on observation of clinical signs and nephropathy, respectively. As nephrotoxicity and accumulation of hyaline droplets were observed in male rats at all dose levels, no NOAEL for male rats was identified.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
dog
Quality of whole database:
Read-across from the analogue substance d-limonene for which there are subacute and subchronic studies in rats, mice and dogs.
System:
urinary
Organ:
kidney

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

There was no data on l-limonene repeated dose toxicity, therefore a read-across approach with the structural analogue d-limonene was followed. For further information on read-across justification, see section 13: point "read-across approach"

In summary, the following NOAEL were found by gavage administration of d-limonene:

 

Study

Species

Sex

NOAEL

LOAEL

16-day oral
(NTP, 1990)

rat

Male

825 mg/kg bw/d

1650 mg/kg bw/d
(decreased bw gain)

16-day oral
(NTP, 1990)

rat

Female

1650 mg/kg bw/d

3300 mg/kg bw/d
(decreased bw gain)

16-day oral

(NTP, 1990)

mouse

Male

1650 mg/kg bw/d

3300 mg/kg bw/d
(increased mortality)

16-day oral
(NTP, 1990)

mouse

Female

1650 mg/kg bw/d

3300 mg/kg bw/d
(increased mortality)

90-day oral
(NTP, 1990)

rat

Male

600 mg/kg bw/d*

1200 mg/kg bw/d
(decreased bw gain)

90-day oral
(NTP, 1990)

rat

Female

600 mg/kg bw/d

1200 mg/kg bw/d
(clinical signs)

90-day oral
(NTP, 1990)

mouse

Male

500 mg/kg bw/d

1000 mg/kg bw/d
(decreased bw gain and clinical signs)

90-day oral
(NTP, 1990)

mouse

Female

500 mg/kg bw/d

1000 mg/kg bw/d
(clinical signs)

180-day oral
(Webb, 1990)

dog

Male

100 mg/kg bw/d

1000 mg/kg bw/d
(increased relative kidney weight)

180-day oral
(Webb, 1990)

dog

Female

100 mg/kg bw/d

1000 mg/kg bw/d
(increased relative and absolute kidney weights)

180-day oral
(Tsuji, 1975)

dog

Male

1000 mg/kg bw/d

3000 mg/kg bw/d
(decreased bw gain and protein casts observed in the renal tubule)

180-day oral
(Tsuji, 1975)

dog

Female

340 mg/kg bw/d

1000 mg/kg bw/d
(decreased bw gain and protein casts observed in the renal tubule)

* Signs of nephrotoxicity were observed in all treated males. They were not presented in this table, as they are known to be male-rat specific and not relevant for humans.  Reference: Meek M.E. et al. (2003) A Framework for Human Relevance Analysis of Information on Carcinogenic Modes of Action, Critical Reviews in Toxicology, 33(6): 591 -653.  

 

When administered orally by gavage for at least 6 months, d-limonene induces effect from 1000 mg/kg bw/d. At this dose level, following 90 days of exposure, d-limonene induces decreased bodyweight gain and clinical signs in mouse. 180 days of exposure to d-limonene at this dose level decreased bodyweight gain and increased relative and absolute kidney weights (with protein casts in the renal tubules of females) in dogs.

The key value has been then selected to be the LOAEL at 1000 mg/kg bw/d. The key study was then selected to be the 180-d toxicity study by oral route in dogs (Webb, 1990) for DNEL derivation since mammalian exposure is more relevant than rodent exposure regarding human health effect assessment. Moreover effects were seen in this study on the target organ of d-limonene, kidneys.

Some additional studies, which were designed to elicit the nephrotoxicity of d-limonene in male rats are also presented:

 

Study

Species

Sex

NOAEL

LOAEL

90-day: mechanism for renal toxicity (Webb, 1989)

rat

Male

5mg/kg bw/d

150 mg/kg bw/d

26-day: mechanism for renal toxicity (Kanerva, 1987a)

rat

Male

#

75 mg/kg bw/d

90-day: mechanism for renal toxicity (Kanerva, 1987b)

rat

Male

#

150 mg/kg bw/d

#: No NOAEL could be found

 Repeated dose toxicity: via oral route - systemic effects (target organ) urogenital: kidneys

Justification for classification or non-classification

Harmonized classification:

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

Self classification:

l-limonene should not be classified for repeated dose toxicity, as the threshold value for effect is 1000 mg/kg bw/d in animal following 90 days of exposure.

CLP Annex I, 3.9.2.8.1. (e) also states that d-limonene induces a male-rat specific effect on kidney, which is not deemed relevant for classification purpose. Therefore l-limonene should not be classified for repeated toxicity.