Registration Dossier

Administrative data

Description of key information

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 day 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 (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, which was the lowest dose tested with no effect.

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:
experimental study
Adequacy of study:
key study
Study period:
From September 18 to October 04, 1979
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
GLP study performed similarly to OECD Guideline 407 but with deviations: study performed only for 16 days; dosing 5 days/week instead of 7 days/week; food consumption, haematological and clinical biochemical test not followed
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
study performed only for 16 days; dosing 5 days/week instead of 7 days/week; food consumption, haematological and clinical biochemical test not followed
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: F344/N
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories (Portage, USA)
- Age at study initiation: 6-7 weeks
- Weight at study initiation: Males: 109-117 g; females: 97-103 g
- Housing: Housed in groups of five in polycarbonate cages
- Diet (e.g. ad libitum): Purina Lab Blox (Chesapeake Feed Co., Beltsville, USA), ad libitum
- Water (e.g. ad libitum): Automatic watering system (Edstrom Industries, Waterford, USA), ad libitum
- Acclimation period: 12 or 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 64-76 °F
- Humidity (%): 80-90%
- 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 oral 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:
not specified
Details on analytical verification of doses or concentrations:
Not applicable
Duration of treatment / exposure:
12 doses over 16 days
Frequency of treatment:
Once per day; 5 days/week
Remarks:
Doses / Concentrations:
0, 413, 825, 1650, 3300 and 6600 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
Five
Control animals:
yes, concurrent vehicle
Details on study design:
- 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: Initially and once per week thereafter
Sacrifice and pathology:
GROSS PATHOLOGY: Yes; necropsy performed on all animals
HISTOPATHOLOGY: Yes; performed on seven rats from survivors of highest dose groups
Other examinations:
No
Statistics:
No data
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No treatment-related clinical signs were observed in rats that received doses of 1650 mg/kg bw/day or lower.
Mortality:
mortality observed, treatment-related
Description (incidence):
All rats in 6600 mg/kg bw/day group and 5/5 males and 3/5 females in 3300 mg/kg bw/day group died within the first 2 days.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Final mean body weight of male rats that received 1650 mg/kg bw/day was 10% lower than that of the vehicle controls.
- Final mean body weight of female rats that received 3300 mg/kg bw/day was 8% lower than that of the vehicle controls.
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
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:
no effects observed
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
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
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
Critical effects observed:
no

Table 1: Survival and mean body weights of rats in the 16-day gavage studies of d-limonene

 

Dose (mg/kg bw/day)

Survival (a)

Mean body weights (grams)

Final weight relative
to vehicle controls (%)

Initial (b)

Final

Change (c)

Male

0

  5/5

115 ± 2

173 ± 3

58 ± 3

-

413

  5/5

113 ± 2

171 ± 4

58 ± 3

99

825

  5/5

113 ± 3

173 ± 4

60 ± 5

100

1650

  5/5

113 ± 3

156 ± 4

43 ± 3

90

3300

 (d) 0/5

114 ± 2

(e)

(e)

(e)

6600

 (f) 0/5

111 ± 2

(e)

(e)

(e)

Female

0

  5/5

98 ± 1

123 ± 1

25 ± 1

-

413

  5/5

101 ± 2

(g) 139 ± 2

38 ± 3

113

825

  5/5

100 ± 2

131 ± 3

31 ± 4

107

1650

  5/5

101 ± 2

127 ± 1

27 ± 2

103

3300

 (f) 2/5

102 ± 2

113 ± 8

10 ± 5

92

6600

 (f) 0/5

103 ± 4

(e)

(e)

(e)

(a) Number surviving/number initially in the group

(b) Initial group mean body weight ± standard error of the mean. Subsequent calculations are based on animals surviving to the end of the study.

(c) Mean body weight change of the survivors ± standard error of the mean

(d)Day of death: 1, 1, 1, 1, 2

(e) No data are reported due to the 100% mortality in this group.

(f) Day of death all 1

(g) One final body weight not recorded; weight change based on remaining four animals.

Conclusions:
The NOAEL for male and female rats were considered to be 825 and 1650 mg/kg bw/day, respectively. The LOAEL for male and female rats were considered to be 1650 and 3300 mg/kg bw/day, respectively, based on decreased bodyweight gains.
Executive summary:

In a 16-day subacute toxicity study performed similarly to OECD Guideline 407 and in compliance with GLP, d-limonene 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). 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 seven rats from survivors of highest dose groups.

 

All rats that received 6600 mg/kg bw/day and 5/5 males and 3/5 females that received 3300 mg/kg bw/day of 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.

 

Therefore, the NOAEL for male and female rats were considered to be 825 and 1650 mg/kg bw/day, respectively. The LOAEL for male and female rats were considered to be 1650 and 3300 mg/kg bw/day, respectively, based on decreased bodyweight gains.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From September 17 to October 03, 1979
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 407 but with deviations: study performed only for 16 days; dosing 5 days/week instead of 7 days/week; food consumption, haematological and clinical biochemical test not followed
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
study performed only for 16 days; dosing 5 days/week instead of 7 days/week; food consumption, haematological and clinical biochemical test not followed
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Limit test:
no
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories (Portage, USA)
- Age at study initiation: 6-8 weeks
- Weight at study initiation: Males: 24.4-26.2 g; females: 19.6-21.9 g
- Housing: Housed in groups of five in polycarbonate cages
- Diet (e.g. ad libitum): Purina Lab Blox (Chesapeake Feed Co., Beltsville, USA), ad libitum
- Water (e.g. ad libitum): Automatic watering system (Edstrom Industries, Waterford, USA), ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 64-76 °F
- Humidity (%): 80-90%
- 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 oral 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:
not specified
Details on analytical verification of doses or concentrations:
Not applicable
Duration of treatment / exposure:
12 doses over 16 days
Frequency of treatment:
Once per day; 5 days/week
Remarks:
Doses / Concentrations:
0, 413, 825, 1650, 3300 and 6600 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
Five
Control animals:
yes, concurrent vehicle
Details on study design:
- 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: Initially and once per week thereafter
Sacrifice and pathology:
GROSS PATHOLOGY: Yes; necropsy performed on all animals
HISTOPATHOLOGY: Yes; performed on six mice from survivors of highest dose groups
Other examinations:
No
Statistics:
No data
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No compound-related clinical signs were observed in mice that received 1650 mg/kg bw/day and lived to the end of the studies.
Mortality:
mortality observed, treatment-related
Description (incidence):
All but one of 20 mice that received 3300 or 6600 mg/kg bw/day died within 3 days.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Vehicle control mice gained little or no weight.
- Slight bodyweight loss was observed in all treated groups, but without any clear dose-response relationship.
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
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:
no effects observed
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
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
Critical effects observed:
no

Table 1: Survival and mean body weights of mice in the 16-day gavage studies of d-limonene

 

Dose (mg/kg bw/day)

Survival (a)

Mean body weights (grams)

Final weight relative to vehicle controls (%)

Initial (b)

Final

Change (c)

Male

0

 5/5

25.4 ± 0.4

26.0 ± 0.9

0.6 ± 0.9

-

413

 5/5

25.2 ± 0.5

23.0 ± 0.9

 -2.2 ± 0.8

88.5

825

 5/5

24.6 ± 0.2

24.2 ± 0.7

-0.4 ± 0.6

93.1

1650

(d) 4/5

25.6 ± 0.6

25.3 ± 1.3

-0.5 ± 1.7

97.3

3300

(e) 1/5

24.8 ± 0.4

19

-5

73.1

6600

(f) 0/5

24.6 ± 0.2

(g)

(g)

(g)

Female

0

 5/5

20.2 ± 0.2

21.8 ± 1.1

 1.6 ± 1.1

-

413

 5/5

21.4 ± 0.5

20.8 ± 0.5

 -0.6 ± 0.5

95.4

825

 5/5

20.0 ± 0.3

19.8 ± 0.6

 -0.2 ± 0.4

90.8

1650

 (h) 4/5

21.2 ± 0.4

22.3 ± 0.6

1.0 ± 1.0

102.3

3300

 (i) 0/5

20.4 ± 0.4

(g)

(g)

(g)

6600

 (j) 0/5

19.8 ± 0.2

(g)

(g)

(g)

(a) Number surviving/number initially in group

(b) Initial group mean body weight ± standard error of the mean. Subsequent calculations are based on animals surviving to the end of the study.

(c) Mean body weight change of the survivors ± standard error of the mean

(d) Day of death: 2

(e) Day of death: 2, 2, 3, 3

(f) Day of death: all 1

(g) No data are reported due to the 100% mortality in this group.

(h) Death due to gavage error

(i) Day of death: 1, 2, 2, 2, 2

(j) Day of death: 1, 1, 1, 2, 2

Conclusions:
The NOAEL in mice was considered to be 1650 mg/kg bw/day, based on mortality rates. The LOAEL for male and female mice were considered to be 3300 mg/kg bw/day, based on increased mortality rates.
Executive summary:

In a 16-day subacute toxicity study performed similarly to OECD Guideline 407 and in compliance with GLP, d-limonene 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.

 

Therefore, the NOAEL in mice was considered to be 1650 mg/kg bw/day, based on mortality rates. The LOAEL for male and female mice were considered to be 3300 mg/kg bw/day, based on increased mortality rates.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From January 28 to April 30, 1980
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 408 but with deviations: dosing 5 days/week instead of 7 days/week; food consumption, haematological and clinical biochemical test not followed
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
dosing 5 days/week instead of 7 days/week; food consumption, haematological and clinical biochemical test not followed
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: F344/N
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories (Portage, USA)
- Age at study initiation: 7-8 weeks
- Weight at study initiation: Males: 136-153 g; females: 101-120 g
- Housing: Housed in groups of five in polycarbonate cages
- Diet (e.g. ad libitum): Purina Lab Blox (Chesapeake Feed Co., Beltsville, USA) or 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: 18 or 19 days

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 60-82 °F
- Humidity (%): 35-80%
- 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 oral 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: Once
- Results: 101-109% of the target concentrations
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Once per day; 5 days/week
Remarks:
Doses / Concentrations:
0, 150, 300, 600, 1200 and 2400 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses were selected based on the mortalities observed at 3300 and 6600 mg/kg bw/day during a 16 day subacute toxicity 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: Initially and once per week thereafter
Sacrifice and pathology:
GROSS PATHOLOGY: Yes; necropsy performed on all animals
HISTOPATHOLOGY: Yes; performed on all vehicle control and high dose animals and all female rats in the 1200 mg/kg bw/day group. Tissues examined include: adrenal glands, brain, colon, esophagus, eyes (if grossly abnormal), femur or sternebrae or vertebrae including marrow, gross lesions and tissue masses with regional lymph nodes, heart, kidneys, liver, lungs and mainstem bronchi, mammary gland, mandibular or mesenteric lymph nodes, pancreas, parathyroids, pituitary gland, prostate/testes or ovaries/uterus, salivary glands, small intestine, spinal cord (if neurologic signs present), spleen, stomach, thymus, thyroid gland, trachea, and urinary bladder. Kidneys examined for all male rats.
Other examinations:
No
Statistics:
No data
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Rough hair coats, lethargy and excessive lacrimation were observed for rats that received 1200 or 2400 mg/kg bw/day.
Mortality:
mortality observed, treatment-related
Description (incidence):
5/10 males and 9/10 females at 2400 mg/kg bw/day died during week 1.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- 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.
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
Description (incidence and severity):
- 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.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Key result
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
not determinable due to adverse toxic effects at highest dose / concentration tested
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:
body weight and weight gain
Remarks on result:
other: if male-rat specific nephrotoxicity (not relevant for humans) is not considered
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:
clinical signs
mortality
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
150 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no

Table 1. Survival and mean body weights of rats in the 13-week gavage studies of d-limonene

 

Dose (mg/kg bw/day)

Survival (a)

Mean body weights (g)

Final weight relative to vehicle controls (%)

Initial (b)

Final

Change (c)

Male

0

10/10

 144 ± 2 

 333 ± 6 

 +189 ± 5 

-

150

10/10

 145 ± 3 

 332 ± 4 

 +187 ± 3

100

300

10/10

 149 ±2 

 330 ± 3 

 +181 ± 4

99

600

10/10

 148 ± 2 

 314± 5 

 +166 ± 5

94

1200

10/10

 139 ± 3 

 292 ± 5 

 +153 ± 6

88

2400

(d) 5/10

 150 ±3 

 255 ± 10 

 +103 ± 10 

77

Female

0

10/10

118 ± 2

185 ± 2

+67± 4

-

150

10/10

115 ± 1

186± 2

+71± 2

101

300

10/10

105 ± 4

181 ± 2

+76± 4

98

600

10/10

114 ± 1

184± 2

+70± 1

99

1200

10/10

116 ± 2

182 ± 3

+66± 3

98

2400

(d) 1/10

113 ± 1

164

 + 56

89

(a) Number surviving/number initially in group

(b) Initial group mean body weight ± standard error of the mean. Subsequent calculations are based on animals surviving to the end of the study.

(c) Mean body weight change of the survivors ± standard error of the mean

(d) Week of death: all 1

 

Table 2. Severity of kidney lesions in male rats in the 13-week gavage study of d-limonene (a)

 

Lesion 

Dose (mg/kg)

 Vehicle Control 

 150 

 300 

 600 

1200

2400

 Regeneration

 (b) 0.8 

 2.4 

 2.5 

 2.5 

 3.7 

0.9

Granular casts

 0 

 1.6 

 2.4 

 2.7 

 3.5 

 0.3 

(a) Severity grades: 1 = minimal; 2 = mild; 3 = moderate; 4 = marked

(b) Average severity grade for all rats in the group

Conclusions:
The NOAEL for male and female rats was considered 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:

In a 13-week subchronic toxicity study performed similarly to OECD Guideline 408 and in compliance with GLP, d-limonene 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). 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.

 

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.

 

Therefore, the NOAEL 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 had primarily been identified in this study.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From January 28 to April 30, 1980
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 408 but with deviations: dosing 5 days/week instead of 7 days/week; food consumption, haematological and clinical biochemical test not followed
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
dosing 5 days/week instead of 7 days/week; food consumption, haematological and clinical biochemical test not followed
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Limit test:
no
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories (Portage, USA)
- Age at study initiation: 7-9 weeks
- Weight at study initiation: Males: 23.8-29.5 g; females: 20.2-21.5 g
- Housing: Housed in groups of five in polycarbonate cages
- Diet (e.g. ad libitum): Purina Lab Blox (Chesapeake Feed Co., Beltsville, USA) or 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: 18 days

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 60-82 °F
- Humidity (%): 35-80%
- 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 oral 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: Once
- Results: 101-109% of the target concentrations
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Once per day; 5 days/week
Remarks:
Doses / Concentrations:
0, 125, 250, 500, 1000 or 2000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses were selected based on the mortalities observed at 3300 and 6600 mg/kg bw/day during a 16 day subacute toxicity 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: Initially and once per week thereafter
Sacrifice and pathology:
GROSS PATHOLOGY: Yes; necropsy performed on all animals
HISTOPATHOLOGY: Yes; performed on all vehicle control and high dose animals. Tissues examined include: adrenal glands, brain, colon, esophagus, eyes (if grossly abnormal), femur or sternebrae or vertebrae including marrow, gallbladder, gross lesions and tissue masses with regional lymph nodes, heart, kidneys, liver, lungs and mainstem bronchi, mammary gland, mandibular or mesenteric lymph nodes, pancreas, parathyroids, pituitary gland, prostate/testes or ovaries/uterus, salivary glands, small intestine, spinal cord (if neurologic signs present), spleen, stomach, thymus, thyroid gland, trachea and urinary bladder.
Other examinations:
No
Statistics:
No data
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Rough hair coats and decreased activity were observed at 1000 and 2000 mg/kg bw/day.
Mortality:
mortality observed, treatment-related
Description (incidence):
- 1/10 male and 2/10 females died at 2000 mg/kg bw/day
- 1/10 female died at 500 mg/kg bw/day
- Several animals in other groups died as a result of gavage error.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
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.
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, non-treatment-related
Description (incidence and severity):
An alveolar cell adenoma was observed in the lung of 1/10 females that received 2000 mg/kg bw/day.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
Key result
Critical effects observed:
no

Table 1. Survival and mean body weights of mice in the 13-week gavage studies of d-limonene

 

Dose (mg/kg bw/day)

Survival (a)

Mean body weights (grams)

Final weight relative to vehicle controls (%)

Initial (b)

Final

Change (c)

Male

0

10/10

 26.6 ± 1.0 

 37.1 ± 1.0 

 +10.5 ± 1.3 

-

125

10/10

 28.8 ± 0.7 

 37.9 ± 1.1 

 +9.1 ± 0.7 

102.2

250

(d) 9/10

 26.5 ± 0.8 

 33.9 ± 0.8 

 +7.6 ± 0.8 

91.4

500

(d) 7/10

 24.7 ± 0.9 

 34.4 ± 0.9 

 +9.7± 1.1 

92.7

1000

(d) 9/10

 28.2 ± 0.9 

 33.3 ± 0.8 

 +5.1 ± 1.1 

89.8

2000

(e) 9/10

 27.7±0.7 

 33.0 ± 0.8 

 +5.6 ± 0.8 

88.9

Female

0

10/10

 21.3 ± 0.2 

 24.7±0.5 

 +3.4 ±0.4 

-

125

(d) 9/10

 20.6 ± 0.3 

 25.9± 0.5 

 +5.2 ± 0.4 

104.9

250

10/10

 20.7 ± 0.3 

 25.4 ± 0.6 

 +4.7 ± 0.4 

102.8

500

(f) 9/10

 20.9 ± 0.2 

 24.9 ±0.5 

 +4.1 ± 0.4 

100.8

1000

10/10

 20.4 ±0.2 

 24.1 ± 0.7 

 +3.7 ±0.7 

97.6

2000

(g) 8/10

 21.0 ± 0.3 

 24.1 ± 0.4 

 +3.4 ± 0.3 

97.6

(a) Number surviving/number initially in group

(b) Initial group mean body weight f standard error of the mean. Subsequent calculations are based on animals surviving to the end of the study.

(c) Mean body weight change of the survivors ± standard error of the mean

(d) Death due to gavage error

(e) Week of death: 1

(f) Week of death: 5

(g) Week of death: 3,4

Conclusions:
The NOAEL was considered to be 500 mg/kg bw/day. The LOAEL was considered 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:

In a 13-week subchronic toxicity study performed similarly to OECD Guideline 408 and in compliance with GLP, d-limonene 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.

 

Therefore, the NOAEL was considered to be 500 mg/kg bw/day. The LOAEL was considered 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:
experimental study
Adequacy of study:
supporting study
Study period:
1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Remarks:
Study designed to evaluate effects of substance on kidneys of rats: dosing 5 days/week instead of 7 days/week; haematological and clinical biochemical test not followed
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Principles of method if other than guideline:
Study designed to evaluate effects of substance on kidneys of rats: dosing 5 days/week instead of 7 days/week; haematological and clinical biochemical test not followed
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male
Route of administration:
oral: gavage
Vehicle:
corn oil
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
5 days/week
Remarks:
Doses / Concentrations:
0, 2, 5, 10, 30 and 75 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
- At 0, 10 and 75 mg/kg bw/day: 5 or 10 males
- At 2, 5 and 30 mg/kg bw/day: 10 males
Control animals:
yes, concurrent vehicle
Key result
Dose descriptor:
NOAEL
Effect level:
5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: chronic nephrosis and a dose-related trend in the increased relative weights of the kidney and liver were observed at 30 and 75 mg/kg bw/day
Key result
Dose descriptor:
LOAEL
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Critical effects observed:
not specified

Preliminary acute toxicity study:

- An increase in the incidence and severity of hyaline droplets was observed in the kidneys of males only. This histological change was accompanied by a treatment-related increase in alpha 2µ-globulin in males only and a greater accumulation of radioactivity in renal cortex of the male rat compared with that in the females dosed with [14C]d-limonene.

 

Subchronic toxicity study:

- Incidence and type of gross pathological lesions observed at necropsy, the cumulative body-weight gain, feed consumption and feed efficiency for treated males did not differ significantly from those of the control males.

- Linear regression analyses indicated a dose-related trend in the increased relative weights of the kidney and liver at 30 and 75 mg/kg bw/day. Histological examination of kidney tissue confirmed that d-limonene induced changes characterized by hyaline droplets, granular casts at the corticomedullary junction and multiple cortical changes collectively classified as chronic nephrosis.

- At the earliest necropsy, 8 days after the start of the treatment, it was evident that d-limonene exacerbated the hyaline droplets at 10 mg/kg bw/day.

- The no-observable-effect level for these effects was 5 mg/kg bw/day.

Conclusions:
The NOAEL and LOAEL were considered to be 5 and 30 mg/kg bw/day, respectively, based on observation of chronic nephrosis. However, mechanisms and specificity of toxicity of d-limonene on kidneys of male rats and its non relevance for humans are well known.
Executive summary:

In a subchronic toxicity study, d-limonene was administered through gavage to groups of 5 or 10 male Fisher-344 rats/dose mixed in corn oil at dose levels of 0, 2, 5, 10, 30 and 75 mg/kg bw/day for 13 weeks (5 days/week). Animals were observed and weighed daily, and feed consumption was recorded weekly. Rats from selected dose groups received interim necropsies from Days 8-29, while all groups were necropsied at the end of the study. In the preliminary acute toxicity study, d-limonene (200 mg/kg bw; 200 µCi/kg bw in corn oil) was administered to a group of male and female Fischer 344 rats by oral gavage. After 24 hours, an increase in the incidence and severity of hyaline droplets containing alpha-2µ-globulin was observed in the kidneys of males only.

 

In the main study, incidence and type of gross pathological lesions observed at necropsy, the cumulative body-weight gain, feed consumption and feed efficiency for treated males did not differ significantly from those of the control males. Linear regression analyses indicated a dose-related trend in the increased relative weights of the kidney and liver at 30 and 75 mg/kg bw/day. Histological examination of kidney tissue confirmed induction of chronic nephrosis characterized by hyaline droplets, granular casts at the corticomedullary junction and multiple cortical changes. At the earliest necropsy, 8 days after the start of the treatment, it was evident that d-limonene exacerbated the hyaline droplets at the 10 mg/kg body weight dose.

 

Therefore, the NOAEL and LOAEL were considered to be 5 and 30 mg/kg bw/day, respectively. However, mechanisms and specificity of toxicity of d-limonene on kidneys of male rats and its non relevance for humans are well known.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
no data
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: well-conducted study but reported in Japanese language
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
no guideline available
Principles of method if other than guideline:
A combination of rules from OECD Guidelines 409 and 452 were followed (by anticipation as thoses guidelines did not exist when the study was conducted).
d-limonene was administered by gavage to dogs for 6 months. This duration was chosen as usual when developing new medicinal products (d-limonene was explored in this study as a possible gallstone solubiliser).
GLP compliance:
no
Species:
dog
Strain:
other: Japanese beagle
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
not specified
Details on oral exposure:
no data
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
6 months
Frequency of treatment:
once a day
Remarks:
Doses / Concentrations:
0.4, 1.2 and 3.6 mL/kg bw/d equivalent to 340, 1000 and 3000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
3/sex/dose
Control animals:
yes
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Decreased body weight and protein casts observed in the renal tubule at 3000 mg/lg bw/d.
Dose descriptor:
LOAEL
Effect level:
3 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Key result
Dose descriptor:
NOAEL
Effect level:
340
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Decreased body weight and protein casts observed in the renal tubule at 1000 mg/lg bw/d.
Dose descriptor:
LOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Critical effects observed:
not specified

- Clinical signs: frequent vomiting and nausea were caused, which appeared to depend on the dose used.

- Body weight: all males in the high dose group, all females in the intermediate group and 2/3 females in the high dose group lost weight over the 6 month-study. Not dose-related in females.

- Food consumption: not affected by treatment except in the intermediate dose group females. Not dose-related.

- Urinalysis: no treatment-related effects.

- Hematology: no treatment-related findings.

- Biochemistry: total cholesterol and glucose decrease in blood in the high dose group. The initial cholesterol level was recovered at the end of the 6-month treatment period in both sexes (when the level had increased in other control and treated groups).

- Organ weights: the relative to body weight kidney and liver weights were slightly higher in the high dose group males than in other groups.

- Histopathology: at 1.2 and 3.6 mL/kg bw/day, protein casts were observed in the renal tubule of most animals. No remarkable treatment-related change was observed in other organs.

Table 1: Protein casts observed in the renal tubule

  Male Female
  Control 0.4 1.2 3.6 Control 0.4 1.2 3.6
No. animals examined 3 3 3 3 3 2 3 3
Protein casts 1 1 2 3 1 2 3 3
Conclusions:
The NOAEL in this study is 1.2 mL/kg bw/day (equivalent to 1000 mg/kg bw/day) in males and 0.4 mL/kg bw/day (equivalent to 340 mg/kg bw/day) in females on the basis of decreased body weight and protein casts observed in the renal tubule at the next dose level. Food consumption was also decreased in the intermediate dose females.
Executive summary:

d-Limonene was administered to three dogs per sex and per dose group by gavage once per day for 6 months at the dose level of 0, 0.4, 1.2 or 3.6 mL/kg bw/day.

All 6 animals (males and females) from the high dose group except one female and all females in the intermediate dose group lost weight between the first and the last day of study. Food consumption only decreased in the intermediate dose group females. Urinalysis and hematology were not affected by treatment. The glucose and total cholesterol levels in blood decreased in the high dose group males and females when compared to the pre-treatment levels; the total cholesterol level recovered the pre-test level by the end of the 6-month treatment period. The relative kidney and liver weights were slightly higher in the high dose group males than in other groups. A dose-related increased incidence of protein casts were observed in the renal tubule: all males in the high dose group and all females in the intermediate and high dose groups showed this effect.

Therefore, the NOAEL in this study is 1.2 mL/kg bw/day (equivalent to 1000 mg/kg bw/day) in males and 0.4 mL/kg bw/day (equivalent to 340 mg/kg bw/day) in females on the basis of decreased body weight and protein casts observed in the renal tubule at the next dose level.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Study conducted similarly to OECD Guideline 409 with deviations: age of animals > 9 months; no data on initial bodyweights; only two dose levels studied; ophthalmological examination not followed; individual animal data not reported
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity in Non-Rodents)
Deviations:
yes
Remarks:
age of animals > 9 months; no data on initial bodyweights; only two dose levels studied; ophthalmological examination not followed; individual animal data not reported
Principles of method if other than guideline:
Not applicable
GLP compliance:
not specified
Limit test:
no
Species:
dog
Strain:
Beagle
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 10-15 months
- Housing: Housed in runs with outdoor access
- Diet (e.g. ad libitum): Meals provided for only 1 hour
- Water (e.g. ad libitum): Ad libitum
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
VEHICLE
- Amount of vehicle (if gavage): 1.2 mL/kg bw/day
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
Not applicable
Duration of treatment / exposure:
180 days
Frequency of treatment:
Each daily dose was divided into two equal amounts, administered at approximately 9.30 am and 2.30 pm.
Remarks:
Doses / Concentrations:
0, 100 or 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
Five
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Highest daily dose was determined in a pilot 28-day study conducted in 5 male and 5 female beagles. Kidney weights for the d-limonene-treated animals were unaffected, but absolute and relative liver weights were slightly increased. Based on these findings, the 1.2 mL/kg bw/day treatment was chosen.
Positive control:
No
Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily for at least 1 hour after dosing

BODY WEIGHT: Yes
- Time schedule for examinations: At study initiation, weekly during the study and at the time of sacrifice

FOOD CONSUMPTION: Yes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At 2 week pre-study (baseline) and then 1, 3 and 6 months during the study
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- Parameters examined: White cell count, red blood cell count, haemoglobin, haematocrit, platelet count, mean corpuscular volume, mean corpuscular haemoglobin and mean corpuscular haemoglobin concentration

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At 2 week pre-study (baseline) and then 1, 3 and 6 months during the study
- Animals fasted: No data
- Parameters examined: Blood urea nitrogen (BUN), BUN/creatinine, creatinine, aspartate amino transferase, alanine amino transferase, phosphorus, glucose, albumin, total protein, globulins, albumin/globulin, alkaline phosphatase, cholesterol, triglycerides, sodium, potassium, calcium and chloride

URINALYSIS: Yes
- Time schedule for collection of urine: 24-hour urine samples were collected at approximately 2 week pre-study and again at 6 months.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data
- Parameters examined: Colour and appearance, specific gravity, occult blood, protein, pH, glucose, ketones, bilirubin and urobilinogen and urinary sediment
Sacrifice and pathology:
GROSS PATHOLOGY: Yes; each animal anaesthetized with pentobarbital, killed by exsanguination and observed for gross post-mortem examinations
HISTOPATHOLOGY: Yes; samples of the following tissues were collected and fixed in 10% buffered formalin for routine histological processing, and light microscopical evaluation of haematoxylin and eosin stained sections: lungs, bronchial lymph node, heart, thoracic aorta, tongue, oesophagus, trachea, thyroid, parathyroid, submandibular lymph node, mesenteric lymph node, stomach, parotid salivary gland, palatine tonsil, liver, gall bladder, duodenum, jejunum, ileum, colon, rectum, urinary bladder, kidneys, testicles with epididymis, prostate, ovaries, uterus, vagina, cervix, adrenals, thymus, psoas muscle, spleen, pancreas, bone/marrow, skin, brain, spinal cord, sciatic nerve, pituitary gland, and eyes. Mallory-Heidenhain-stained kidney sections were also prepared and evaluated for protein accumulation. Kidney weights were determined immediately on removal from the animal. Absolute organ weights were calculated as percentages of the body weights (relative weights).
Other examinations:
None
Statistics:
- Statistically significant differences (P < 0.05, two-sided risk level) were established using least significant difference criteria, provided that Bartlett's test of homogeneity of variance was nonsignificant.
- Dose-response data were also analysed by linear regression (Dixon and Massey, 1969).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Excretion of soft faeces; dose-related occasional mild discomfort during defaecation (presumably because of perianal contact with unabsorbed d-limonene as it passed with the faeces); sporadic episodes of emesis and diarrhoea
Mortality:
no mortality observed
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
Description (incidence and severity):
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.
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
Description (incidence and severity):
Linear regression analyses indicated a positive dose-related trend for absolute and relative female kidney weight and relative male kidney weight.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
Key result
Critical effects observed:
no

Table 1. Organ and bodyweight data for dogs after 6 months daily administration of d-limonene

 

 

 Dose (mg/kg bw/day) 

 

 Control 

 100 

 1000

Male

 Final body weight (kg) 

 11.381 ± 0.828 

 10.889 ± 0.595 

 11.008 ± 0.688 

 Kidney weight (g) 

 57.09 ± 6.02 

 64.56 ± 6.60 

 73.33 ± 8.91 

 Kidney/body weight (%) 

 0.498 ± 0.023 

 0.588 ± 0.035 

 0.661 ± 0.61*

Female

 Final body weight (kg) 

 9.158 ± 0.789 

 9.513 ± 0.315 

 9.176 ± 0.823 

 Kidney weight (g) 

 42.18 ± 3.48 

 45.61 ± 2.29 

 55.36 ±2.58* 

 Kidney/body weight (%) 

 0.461 ± 0.006 

 0.479 ± 0.016 

 0.614 ± 0.032* 

* Statistical significance at P < 0.05,

Values are means ± SEM for 5 dogs.

Conclusions:
The NOAEL and LOAEL for beagle dogs were considered to 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:

In a 6-month subchronic toxicity study performed similarly to OECD Guideline 409, d-limonene 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. Animals were observed daily and weighed at study initiation, weekly during the study and at the time of sacrifice. Feed consumptions were determined throughout the study and blood samples were obtained at 2 week pre-study (baseline) and then 1, 3 and 6 months during the study. At termination all animals were subjected to gross necropsy during which weights of kidneys were recorded and several tissues were processed for microscopical evaluation of haematoxylin and eosin stained sections. Mallory-Heidenhain-stained kidney sections were also prepared and evaluated for protein accumulation.

 

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.

 

Therefore, the NOAEL and LOAEL for beagle dogs were considered to 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:
experimental study
Adequacy of study:
supporting study
Study period:
1987
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
Study designed to evaluate effects of substance on kidneys of rats; dosing 5 days/week instead of 7 days/week; haematological and clinical biochemical test not followed; only liver and kidneys were weighed and processed for histological examinations
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Principles of method if other than guideline:
Subacute toxicity study (1 or 4 weeks). Study designed to evaluate effects of substance on kidneys of rats; dosing 5 days/week instead of 7 days/week; haematological and clinical biochemical test not followed; only liver and kidneys were weighed and processed for histological examinations
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male
Route of administration:
oral: gavage
Vehicle:
corn oil
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
5 or 26 days (5 days/week)
Frequency of treatment:
5 days/week
Remarks:
Doses / Concentrations:
0, 75, 150 or 300 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
Five males
Control animals:
yes, concurrent vehicle
Key result
Dose descriptor:
NOAEL
Based on:
test mat.
Sex:
male
Basis for effect level:
other: nephrotoxicity and accumulation of hyaline droplets (male rat specific)
Remarks on result:
not determinable
Remarks:
no NOAEL identified
Critical effects observed:
not specified

- Neither daily in-life observation nor examination of animals at necropsy revealed any grossly evident indication of dose-related toxicity.

- Weight gains and feed consumption values for treatment groups were, at both time points, similar to those of the vehicle control groups.

- Relative liver and kidney weights in 300 mg/kg bw/day group were significantly higher than the control in animals killed on Days 6 and 27.

- Dose-related hyaline droplet formation associated with renal accumulation of α-globulin was observed in all rats killed on Day 6.

- Chronic nephrosis, characterised by granular casts in the outer zone of the medulla and multiple cortical changes, was observed in the kidneys of rats killed on Day 27.

Conclusions:
As nephrotoxicity and accumulation of hyaline droplets containing α2µ-globulin were observed in male rats (mechanism known to be not relevant for humans) at all dose levels, no NOAEL could be identified in this study. However, mechanisms and specificity of toxicity of d-limonene on kidney of male rats and its non relevance for humans are well known.
Executive summary:

In a subacute toxicity study, d-limonene was administered through gavage to groups of 5 male Fisher-344 rats/dose mixed in corn oil at dose levels of 0, 75, 150 and 300 mg/kg bw/day for 1 or 4 weeks (5 days/week). Animals were weighed daily and feed consumption was recorded weekly. On Days 6 and 27, approximately 24 hours after the 5thand 20thdoses, respectively, animals from the appropriate groups were subjected to gross necropsy during which weights of liver and kidneys were recorded and transverse sections were processed for histological examination. Tissues from right kidney of animals killed on Day 6 were processed for two-dimensional gel electrophoretic evaluation.

Neither daily in-life observation nor examination of animals at necropsy revealed any grossly evident indication of dose-related toxicity. Weight gains and feed consumption values for treatment groups were similar to those of the vehicle control groups. Relative liver and kidney weights in 300 mg/kg bw/day group were significantly higher than the control in animals killed on Days 6 and 27. Dose-related hyaline droplet formation associated with renal accumulation of α2µ-globulin was observed in all rats killed on Day 6. Chronic nephrosis, characterised by granular casts in the outer zone of the medulla and multiple cortical changes, was observed in the kidneys of rats killed on Day 27.

As nephrotoxicity and accumulation of hyaline droplets containing α-globulin were observed in male rats at all dose levels, no NOAEL could be identified in this study. However, mechanisms and specificity of toxicity of d-limonene on kidney of male rats and its non relevance for humans are well known.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
no data
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
Test method and results not sufficiently detailed. Study designed for the assessment of kidney effects of d-limonene
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Principles of method if other than guideline:
91-day subchronic toxicity study: d-limonene (0, 150, 300, 600, 1200 and 2400 mg/kg bw/day) was administered orally (gavage) to rats for 91 days and kidney sections of the surviving male rats were processed for histological examination.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
not specified
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
not specified
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
91 days
Frequency of treatment:
Once daily
Remarks:
Doses / Concentrations:
0, 150, 300, 600, 1200 and 2400 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
- At 0 and 1200 mg/kg bw/day: 10 males and 5 females
- At 150-600 mg/kg bw/day: 10 males
- At 2400 mg/kg bw/day: 5 males and 1 female
Control animals:
yes, concurrent vehicle
Dose descriptor:
NOAEL
Basis for effect level:
other: chronic nephrosis and granular cast formation were observed at all dose levels
Remarks on result:
not determinable
Remarks:
no NOAEL identified
Critical effects observed:
not specified

- Bodyweight gain: Absolute mean bodyweight gain at 600, 1200 and 2400 mg/kg bw/day decreased by 12, 19 and 46%, respectively, relative to controls.

- Dose-related increase in the severity of chronic nephrosis and granular cast formation were observed in male rats at concentrations ranging from 150 to 1200 mg/kg bw/day. At 2400 mg/kg bw/day, the severity of chronic nephrosis was similar to that at 150 mg/kg bw/day and formation of granular cast was observed in 1/10 male.

- Chronic nephrosis was characterised by cytoplasmic basophilia of PCT epithelial cells, tubular hyperplasia or atrophy, fibrosis of Bowman’s capsule and an interstitial fibrolymphocytic response.

- Chronic nephrosis was much more severe in the kidneys of all the male rats treated with d-limonene than in the controls.

- No lesions were observed in kidneys of female rats.

Conclusions:
As chronic nephrosis and granular casts formation were observed in male rats (mechanism known to be not relevant for humans) at all dose levels, no NOAEL could be identified in this study. However, mechanisms and specificity of toxicity of d-limonene on kidney of male rats and its non relevance for humans are well known.
Executive summary:

In a subchronic toxicity study, d-limonene was administered through gavage to groups of male and female rats at dose levels of 0, 150, 300, 600, 1200 and 2400 mg/kg bw/day for 91 days. Surviving animals from the appropriate groups were subjected to gross necropsy and transverse sections of right and left kidneys were processed for histological examination.

Absolute mean bodyweight gain at 600, 1200 and 2400 mg/kg bw/day decreased by 12, 19 and 46%, respectively, relative to controls. Dose-related increase in the severity of chronic nephrosis and granular cast formation were observed in male rats at concentrations ranging from 150 to 1200 mg/kg bw/day. At 2400 mg/kg bw/day, the severity of chronic nephrosis was similar to that at 150 mg/kg bw/day and formation of granular cast was observed in 1/10 male. Treatment-related lesions were not observed in kidneys of female rats.

As chronic nephrosis and granular casts formation were observed in male rats at all dose levels, no NOAEL could be identified in this study. However, mechanisms and specificity of toxicity of d-limonene on kidney of male rats and its non relevance for humans are well known.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
dog

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

To sum up, 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 effects from 1000 mg/kg bw/d. At this dose level, following 90 day 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 (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

 

Justification for classification or non-classification

Substance should not be classified for repeated dose toxicity, as the threshold value for effects is 1000 mg/kg bw/d in animals 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 kidneys, which is not deemed relevant for classification purpose.