Orange, sweet, ext.

Endpoint summary

Description of key information

Repeated dose toxicity: oral
Weight of evidence approach
6-month repeated dose toxicity study in dogs: LOAEL 1000 mg/kg bw/day
90-day repeated dose toxicity study in mice: LOAEL 1000 mg/kg bw/day
90-day repeated dose toxicity study in rats: LOAEL 1200 mg/kg bw/day
supporting 6-month repeated dose toxicity study in dogs: LOAEL 1000 mg/kg bw/day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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:

Remarks:

well-conducted study but reported in Japanese language

Reason / purpose for cross-reference:

reference to same 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

Dose / conc.:

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

Dose / conc.:

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

Remarks:

Original value: 0.4 mL/kg bw/d equivalent to 340 mg/kg bw/day

Dose / conc.:

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

Remarks:

Original value: 1.2 mL/kg bw/d equivalent to 1000 mg/kg bw/day

Dose / conc.:

3 000 mg/kg bw/day (actual dose received)

Remarks:

Original value: 3.6 mL/kg bw/d equivalent to 3000 mg/kg bw/day

No. of animals per sex per dose:

3/sex/dose

Control animals:

yes

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

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:

Three dogs per sex and per dose group were administered d-limonene 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.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

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 for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study 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 or test system 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.

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

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:

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

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

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

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Dose descriptor:

NOAEL

Effect level:

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

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

Key result

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: increased absolute and relative female kidney weight and relative male kidney weight

Critical effects observed:

not specified

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:

Under the test conditions, 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.

 

Under the test conditions, 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.

Reference 3

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From January 28 to April 30, 1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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 for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study 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 or test system 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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

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

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, 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

Dose descriptor:

NOAEL

Effect level:

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

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 (actual dose received)

Sex:

male/female

Basis for effect level:

other: decreased bodyweight gain and occurence of clinical signs of toxicity (rough hair coats and decreased activity)

Critical effects observed:

not specified

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:

Under the test conditions, 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.

 

Under the test conditions, 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.

Reference 4

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From January 28 to April 30, 1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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 for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study 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 or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories (Portage, USA)
- Age at study initiation: 7-8 weeks
- Weight at study initiation: Males: 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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

1 200 mg/kg bw/day (actual dose received)

Dose / conc.:

2 400 mg/kg bw/day (actual dose received)

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

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

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

Details on results:

MORTALITY:
- 5/10 males and 9/10 females at 2400 mg/kg bw/day died during week 1.

CLINICAL SIGNS:
- Rough hair coats, lethargy and excessive lacrimation were observed for rats that received 1200 or 2400 mg/kg bw/day.

BODY WEIGHT AND WEIGHT GAIN
- 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.

HISTOPATHOLOGY
- 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.

Dose descriptor:

NOAEL

Effect level:

>= 150 - <= 2 400 mg/kg bw/day (actual dose received)

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

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;

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

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

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

Critical effects observed:

not specified

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:

Under the test conditions, 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.

 

Under the test conditions, 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.

Reference 5

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:

study well documented, meets generally accepted scientific principles, acceptable 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

Reason / purpose for cross-reference:

reference to same 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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

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

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

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:

Under the test conditions, the NOAEL and LOAEL were considered to be 5 and 30 mg/kg bw/day, respectively, based on observation of chronic nephrosis. 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.

 

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

Reference 6

Endpoint:

sub-chronic toxicity: oral

Type of information:

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

Adequacy of study:

supporting study

Justification for type of information:

See attached justification

Reason / purpose for cross-reference:

read-across source

Sex:

male/female

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

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. This result was read-across to orange oil.

Executive summary:

Three dogs per sex and per dose group were administered d-limonene 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. This result was read-across to orange oil.

Reference 7

Endpoint:

sub-chronic toxicity: oral

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

See attached justification

Reason / purpose for cross-reference:

read-across source

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

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:

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

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

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

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY: 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

BODY WEIGHT AND WEIGHT GAIN: No effects

FOOD CONSUMPTION: No effects

HAEMATOLOGY/CLINICAL CHEMISTRY/URINALYSIS: 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.

ORGAN WEIGHTS: Linear regression analyses indicated a positive dose-related trend for absolute and relative female kidney weight and relative male kidney weight.

GROSS PATHOLOGY/HISTOPATHOLOGY: No effects

Dose descriptor:

NOAEL

Effect level:

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

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

Key result

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: increased absolute and relative female kidney weight and relative male kidney weight

Critical effects observed:

not specified

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:

Under the test conditions, 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. This result was read-across to orange oil.

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.

 

Under the test conditions, 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. This result was read-across to orange oil.

Reference 8

Endpoint:

sub-chronic toxicity: oral

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

See attached justification

Reason / purpose for cross-reference:

read-across source

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- 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

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, 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

Dose descriptor:

NOAEL

Effect level:

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

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 (actual dose received)

Sex:

male/female

Basis for effect level:

other: decreased bodyweight gain and occurence of clinical signs of toxicity (rough hair coats and decreased activity)

Critical effects observed:

not specified

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:

Under the test conditions, 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. This result was read-across to orange oil.

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.

 

Under the test conditions, 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. This result was read-across to orange oil.

Reference 9

Endpoint:

sub-chronic toxicity: oral

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

See attached justification

Reason / purpose for cross-reference:

read-across source

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- 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

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

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

Dose descriptor:

NOAEL

Effect level:

> 150 - <= 2 400 mg/kg bw/day (actual dose received)

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

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;

Dose descriptor:

LOAEL

Effect level:

1 200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: occurrence of clinical signs of toxicity (rough hair coats, lethargy and excessive lacrimation)

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

Dose descriptor:

LOAEL

Effect level:

1 200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: occurrence of clinical signs of toxicity (rough hair coats, lethargy and excessive lacrimation)

Critical effects observed:

not specified

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:

Under the test conditions, 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. This result was read-across to orange oil.

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.

 

Under the test conditions, 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. This result was read-across to orange oil.

Reference 10

Endpoint:

sub-chronic toxicity: oral

Type of information:

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

Adequacy of study:

supporting study

Justification for type of information:

See attached justification

Reason / purpose for cross-reference:

read-across source

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

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:

Under the test conditions, the NOAEL and LOAEL were considered to be 5 and 30 mg/kg bw/day, respectively, based on observation of chronic nephrosis. Mechanisms and specificity of toxicity of d-limonene on kidneys of male rats and its non relevance for humans are well known. This result was read-across to orange oil.

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.

 

Under the test conditions, the NOAEL and LOAEL were considered to be 5 and 30 mg/kg bw/day, respectively, but mechanisms and specificity of toxicity of d-limonene on kidneys of male rats and its non relevance for humans are well known. This result was read-across to orange oil.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

other: WoE approach

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

A weight of evidence approach was choosen based on the availability of repeated dose toxicity studies in different species for the major constituent of orange oil, limonene. All studies were taken into account in the derivation of the DNEL, because although the most sensitive NOAEL is from the 1-year dog study, it may not be the most appropriate NOAEL to base the DNEL on. In the 90-d mouse study (NTP, 1990), a NOAEL of 500 mg/kg bw/day was observed, while the LOAEL in this study was the same as in the dog study (1000 mg/kg bw/day). As the studies are performed in different species, the choice for the mouse NOAEL of 500 mg/kg bw/day is arbitrary. However, the shared LOAEL can be used as a starting point for the derivation of the DNEL. Furthermore, a 90-day rat study which resulted in a LOAEL of 1200 mg/kg bw/day. An additional dog study is available as supporting study in which a LOAEL of 1000 mg/kg bw/day was observed (NOAEL 340 mg/kg bw/day).

 

The 6 -month subchronic toxicity study in dogs was performed similarly to OECD Guideline 409 (Webb, 1990). 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.

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.

Under the test conditions, 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.

 

The 90-d repeated dose toxicity studies in mice and one in rats were both performed by the National Toxicology Program (Jameson, 1990).

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).

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.

Under the test conditions, 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.

 

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).

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.

Under the test conditions, the NOAEL for female rats was considered to be 600 mg/kg bw/day. 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. 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. 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.

 

An additional 6 -month study in dogs is available in which three dogs per sex and per dose group were administered d-limonene 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.

Under the test conditions, the NOAEL was considered to be 340 mg/kg bw/day. The LOAEL was considered to be 1000 mg/kg bw/day, based on decreased body weight and protein casts observed in the renal tubule in females.

Justification for classification or non-classification

Based on the criteria outlined in 67/548/EEC and 1272/2008/EC orange oil does not have to be classified with regard to repeated toxicity.