Registration Dossier

Administrative data

Description of key information

All studies available show  oral LD50 at least equal to or higher than 2000 mg/kg bw in rats.
All studies available show dermal LD50 equal to or higher than 5000 mg/kg bw in rabbits.

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Referenceopen allclose all

Endpoint:
acute toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
D-Limonene is one of the main constituents of dipentene multiconstituent (REACTION MASS OF BETA-PHELLANDRENE AND D-LIMONENE AND L-LIMONENE). Therefore, data on dipentene multiconstituent can be used for extrapolation to d-limonene. See read-across justification document in section 13.
Reason / purpose:
read-across source
Preliminary study:
Not applicable
Sex:
female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
1/6 on Day 6
Clinical signs:
- Decrease in spontaneous activity (4/6), muscle tone and righting reflex (3/6); increased salivation and piloerection (3/6) on Day 1; animals recovered to normal at 24 hours post dose.
Body weight:
- 7% decrease in body weight on Day 2 of animal that died on Day 6
- Absence of body weight gain on Day 2, recovered a normal body weight on Day 7
Gross pathology:
- White thinning of the corpus of the animal that died on Day 6
- Thickness (5/5) and white coloration (2/5) of forestomach
Other findings:
None

Table 1: Body weight and weight gain in grams

Animals

Day 0

Day 2

Day 2-Day 0

Day 7

Day 7-Day 0

Day 14

Day 14-Day 0

1

213

198

-15

Dead

Dead

Dead

Dead

2

230

237

7

256

26

285

55

3

207

211

4

245

38

249

42

4

219

225

6

241

22

264

45

5

221

222

1

252

31

268

47

6

225

239

14

263

38

272

47

Mean

219.2

222

2.8

251.4

31

267.2

47.2

Standard deviation

8.3

15.6

9.7

8.7

7.1

13

4.8

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The oral LD50 for dipentene was found to be greater than 2000 mg/kg bw in the Sprague-Dawley rats and therefore it is not classified according to Directive 67/548/EEC and CLP Regulation (EC) No 1272/2008.
Executive summary:

In an oral acute toxicity (limit test) study performed in accordance with GLP and OECD guideline 423, groups of six Sprague-Dawley female rats received a single oral (gavage) dose of dipentene at 2000 mg/kg bw. Animals were then observed for mortality, body weight and clinical signs of toxicity for 14 days and were all macroscopically necropsied after sacrifice. The observations were compared to historical control data.

One animal died on Day 6 during the study. A decrease in spontaneous activity (4/6), muscle tone and righting reflex (3/6) and increased salivation and piloerection (3/6) was noted on first day of study. All animals recovered to normal behavior at 24 hours post dose. A 7% decrease in body weight on Day 2 was observed in animal that died on Day 6. In surviving animals absence of body weight gain was noted on Day 2 that recovered normal body weight on Day 7. Thickness (5/5) and white coloration (2/5) of forestomach of surviving animals and white thinning of the corpus of the dead animal was noted on necropsy. The oral LD50 was found to be greater than 2000 mg/kg bw in rats.

The oral LD50 for dipentene was found to be greater than 2000 mg/kg bw in the Sprague-Dawley rats and therefore it is not classified according to Directive 67/548/EEC and CLP Regulation (EC) No 1272 /2008.

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1972
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
Study performed similarly to OECD guideline 401 with deviations: no data about purity of the test substance; no data on source of animals and environmental conditions; bodyweights not recorded
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 401 (Acute Oral Toxicity)
Deviations:
yes
Remarks:
no data about purity and no certificate of analysis of the test substance; no data on source of animals and environmental conditions; bodyweights not recorded
Principles of method if other than guideline:
Not applicable
GLP compliance:
no
Test type:
standard acute method
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 200-250 g
- Fasting period before study: 16 hours
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
Route of administration:
oral: unspecified
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
None
Doses:
5000 mg/kg bw
No. of animals per sex per dose:
10 males
Control animals:
not specified
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Mortality was observed at 1 and 6 hours after dosing and daily thereafter for 14 days
- Necropsy of survivors performed: Yes
Statistics:
No data
Preliminary study:
Not applicable
Key result
Sex:
male
Dose descriptor:
LD50
Effect level:
> 5 000 mg/kg bw
Based on:
test mat.
Mortality:
No mortality was observed.
Clinical signs:
Lethargy
Body weight:
No data
Gross pathology:
No data
Other findings:
None

None

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The oral LD50 for d-limonene is higher than 5000 mg/kg bw in rats therefore it is not classified according to Directive 67/548/EEC and CLP Regulation (EC) No 1272/2008.
Executive summary:

In an acute oral toxicity study performed similarly to OECD Guideline 401, ten male Wistar rats were given a single oral dose of undiluted d-limonene at 5000 mg/kg bw. Animals were then observed for mortality and clinical signs of toxicity for 14 days and were all macroscopically necropsied after sacrifice. The rats experienced lethargy. No deaths occurred.

 

Therefore, the oral LD50 for d-limonene is higher than 5000 mg/kg bw in rats therefore it is not classified according to Directive 67/548/EEC and CLP Regulation (EC) No 1272/2008.

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1975
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Original reference in Japanese language
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Principles of method if other than guideline:
Standard acute method
GLP compliance:
not specified
Test type:
standard acute method
Limit test:
no
Species:
mouse
Strain:
other: ddN
Sex:
male/female
Details on test animals and environmental conditions:
None
Route of administration:
oral: unspecified
Vehicle:
other: 10% gum arabic-water
Details on oral exposure:
VEHICLE
- Concentration in vehicle: 6, 7, 8.5, 10.5, 14, 15, 16.5 and 20 mL/kg bw

DOSAGE PREPARATION (if unusual): Test solution was prepared with 10% gum arabic-water.
Doses:
3000, 3500, 4300, 5300, 7000, 7500, 8300 and 10000 mg/kg bw
No. of animals per sex per dose:
- At 3000 mg/kg bw: 10 males
- At 3500-8300 mg/kg bw: 10 mice/sex
- At 10000 mg/kg bw: 10 females
Control animals:
not specified
Details on study design:
None
Statistics:
No data
Preliminary study:
Not applicable
Key result
Sex:
male
Dose descriptor:
LD50
Effect level:
5 600 mg/kg bw
Based on:
test mat.
95% CL:
>= 4 800 - <= 6 500
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
6 600 mg/kg bw
Based on:
test mat.
95% CL:
>= 5 500 - <= 7 900
Mortality:
- Mortalities (male): 0, 10, 0, 30, 60, 90 and 100% at 3000, 3500, 4300, 5300, 7000, 7500 and 8300 mg/kg bw, respectively.
- Mortalities (female): 0, 20, 20, 30, 60, 90 and 100% at 3500, 4300, 5300, 7000, 7500, 8300 and 10000 mg/kg bw, respectively.
Clinical signs:
No data
Body weight:
No data
Gross pathology:
No data
Other findings:
None

None

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The oral LD50 for d-limonene is higher than 5000 mg/kg bw in mice therefore it is not classified according to Directive 67/548/EEC and CLP Regulation (EC) No 1272/2008.
Executive summary:

In an acute oral toxicity study, groups (10/sex) of mice were given a single oral dose of d-limonene (50%) in 10% gum-arabic-water at 3000, 3500, 4300, 5300, 7000, 7500, 8300 or 10000 mg/kg bw.

 

Mortalities in males were 0, 10, 0, 30, 60, 90 and 100% at 3000, 3500, 4300, 5300, 7000, 7500 and 8300 mg/kg bw, respectively. Mortalities in females were 0, 20, 20, 30, 60, 90 and 100% at 3500, 4300, 5300, 7000, 7500, 8300 and 10000 mg/kg bw, respectively. The oral LD50 in males and females were 5600 (4800-6500) and 6600 (5500-7900) mg/kg bw, respectively.

  

The oral LD50 for d-limonene is higher than 5000 mg/kg bw in mice therefore it is not classified according to Directive 67/548/EEC and CLP Regulation (EC) No 1272/2008.

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1975
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Original reference in Japanese language
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Principles of method if other than guideline:
Standard acute method
GLP compliance:
not specified
Test type:
standard acute method
Limit test:
no
Species:
rat
Strain:
other: Wistar JCL
Sex:
male/female
Details on test animals and environmental conditions:
None
Route of administration:
oral: unspecified
Vehicle:
other: 10% gum arabic-water
Details on oral exposure:
VEHICLE
- Concentration in vehicle: 3.6, 4.6, 6.0, 7.8, 10.2, 13.2, 17.2, 22.4, 29.2 and 37.8 mL/kg bw

DOSAGE PREPARATION (if unusual): Test solution was prepared with 10% gum arabic-water.
Doses:
1500, 1900, 2500, 3300, 4300, 5600, 7300, 9400, 12200 and 15900 mg/kg bw
No. of animals per sex per dose:
10
Control animals:
not specified
Details on study design:
None
Statistics:
No data
Preliminary study:
Not applicable
Key result
Sex:
male
Dose descriptor:
LD50
Effect level:
4 400 mg/kg bw
Based on:
test mat.
95% CL:
>= 3 400 - <= 5 900
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
5 200 mg/kg bw
Based on:
test mat.
95% CL:
>= 3 900 - <= 7 000
Mortality:
- Mortalities (male): 0, 30, 30, 40, 60, 40, 90, 70, 80 and 90% at 1500, 1900, 2500, 3300, 4300, 5600, 7300, 9400, 12200 and 15900 mg/kg bw, respectively.
- Mortalities (female): 0, 20, 50, 40, 50, 60, 60, 90 and 100% at 1500, 1900, 2500, 3300, 4300, 5600, 7300, 9400, 12200 and 15900 mg/kg bw, respectively.
Clinical signs:
No data
Body weight:
No data
Gross pathology:
No data
Other findings:
None

None

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The oral LD50 for d-limonene is higher than 2000 mg/kg bw in rats therefore it is not classified according to Directive 67/548/EEC and CLP Regulation (EC) No 1272/2008.
Executive summary:

In an acute oral toxicity study, groups (10/sex) of Wistar JCL rats were given a single oral dose of d-limonene (50%) in 10% gum-arabic-water at 1500, 1900, 2500, 3300, 4300, 5600, 7300, 9400, 12200 and 15900 mg/kg bw.

 

Mortalities in males were 0, 30, 30, 40, 60, 40, 90, 70, 80 and 90% at 1500, 1900, 2500, 3300, 4300, 5600, 7300, 9400, 12200 and 15900 mg/kg bw, respectively. Mortalities in females were 0, 20, 50, 40, 50, 60, 60, 90 and 100% at 1900, 2500, 3300, 4300, 5600, 7300, 9400, 12200 and 15900 mg/kg bw, respectively. The oral LD50 in males and females were 4400 (3400-5900) and 5200 (3900-7000) mg/kg bw, respectively.

 

The oral LD50 for d-limonene is higher than 2000 mg/kg bw in rats therefore it is not classified according to Directive 67/548/EEC and CLP Regulation (EC) No 1272/2008.

Endpoint:
acute toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
D-Limonene is one of the two isomers of dipentene. Therefore, data on dipentene can be used for extrapolation to d-limonene. See read-across justification document in section 13.
Reason / purpose:
read-across source
Key result
Sex:
male
Dose descriptor:
LD50
Effect level:
5 300 mg/kg bw
Based on:
test mat.
95% CL:
>= 4 600 - <= 6 000
Mortality:
- 1/10 at 3200 mg/kg bw; 3/10 at 4000 mg/kg bw; 6/10 at 5000 mg/kg bw and 7/10 and 6250 mg/kg bw
- Death occurred four hours to overnight following administration of drug
Clinical signs:
Ataxia, loss of righting reflex, slow breathing, lethargy and urinary incontinence

None

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The oral LD50 for dl-limonene was found to be greater than 2000 mg/kg bw in the rats and therefore it is not classified according to Directive 67/548/EEC and CLP Regulation (EC) No 1272/2008.
Executive summary:

In an oral acute toxicity study a group of 10 male albino Wistar rats received oral doses of dl-limonene at 3200, 4000, 5000 and 6250 mg/kg bw. Animals were then observed for mortality and clinical signs of toxicity for 14 days and were all macroscopically necropsied after sacrifice.

One, three, six and seven deaths occurred at each tested dose level of 3200, 4000, 5000 and 6250 mg/kg bw, respectively. Animals experienced ataxia, loss of righting reflex, slow breathing, lethargy and urinary incontinence during the study. The calculated LD50 for dl-limonene was 5300 mg/kg bw with 95% confidence limit of 4600 and 6000 mg/kg bw. 

The oral LD50 for dl-limonene was found to be greater than 2000 mg/kg bw in the rats and therefore it is not classified according to Directive 67/548/EEC and CLP Regulation (EC) No 1272/2008.

Endpoint:
acute toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
L-Limonene is the S-isomer of d-limonene. Therefore, data on l-limonene can be used for extrapolation to d-limonene. See read-across justification document in section 13.
Reason / purpose:
read-across source
Principles of method if other than guideline:
Standard acute method (limit test)
Preliminary study:
Not applicable
Key result
Sex:
not specified
Dose descriptor:
LD50
Effect level:
> 5 000 mg/kg bw
Based on:
test mat.
Mortality:
No mortality was observed.
Clinical signs:
No
Body weight:
No data
Gross pathology:
No data
Other findings:
None

None

Interpretation of results:
not classified
Conclusions:
The oral LD50 for l-limonene is higher than 5000 mg/kg bw in rats therefore it is not classified according to Directive 67/548/EEC and CLP Regulation (EC) No 1272/2008.
Executive summary:

In an acute oral toxicity study, 10 rats were given a single oral dose of l-limonene at 5000 mg/kg bw. Animals were then observed for mortality and clinical signs of toxicity for 14 days. No deaths and clinical signs of toxicity occurred during the observation period.

 

Therefore, the oral LD50 for l-limonene is higher than 5000 mg/kg bw in rats therefore it is not classified according to Directive 67/548/EEC and CLP Regulation (EC) No 1272/2008.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
2 000 mg/kg bw
Quality of whole database:
The key study is the most recent study conducted according to GLP on a read-across substance, supported by old non GLP studies on the registered substance but with consistent results.

Acute toxicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Acute toxicity: via dermal route

Link to relevant study records

Referenceopen allclose all

Endpoint:
acute toxicity: dermal
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1972
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study deemed to have been performed similarly to OECD Guideline 402, but no detailed on test conditions and results: no data on age, gender and source of animals; no data on housing and environmental conditions; observation period: 7 days; performed on abraded skin
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
Deviations:
yes
Remarks:
no data on age, gender and source of animals; no data on housing and environmental conditions; observation period: 7 days; performed on abraded skin
Principles of method if other than guideline:
Not applicable
GLP compliance:
no
Test type:
standard acute method
Limit test:
yes
Species:
rabbit
Strain:
New Zealand White
Sex:
not specified
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 1.9-2.4 kg

Type of coverage:
occlusive
Vehicle:
not specified
Details on dermal exposure:
- Area of exposure: Clipped abraded abdominal skin
- Type of wrap if used: Wrapped with binders of rubber dam, gauze and adhesive tape
Duration of exposure:
24 hours
Doses:
5000 mg/kg bw
No. of animals per sex per dose:
10 rabbits
Control animals:
not specified
Details on study design:
- Duration of observation period following administration: 7 days
- Frequency of observations and weighing: Once daily for 7 days
- Necropsy of survivors performed: Yes
- Other examinations performed: Dermal reactions: erythema, edema and atonia


Statistics:
No data
Preliminary study:
Not applicable
Key result
Sex:
not specified
Dose descriptor:
LD50
Effect level:
> 5 000 mg/kg bw
Based on:
test mat.
Mortality:
No mortality
Clinical signs:
No evidence of toxicity; all animals appeared normal at the end of the study
Body weight:
Normal gain in bodyweight
Gross pathology:
No abnormalities were noted at necropsy.
Other findings:
Dermal reactions:
- Slight erythema in 6, 6, 3 and 1 animals on Days 1, 2, 3 and 4, respectively; moderate erythema in 3 animals on Day 1
- Slight edema in 5, 5, 4 and 1 animals on Days 1, 2, 3, and 4, respectively; moderate edema in 4 and 1 animals on Days 1 and 2
- Complete recovery within 5 days
- No signs of atonia were observed during the study

None

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The acute dermal LD50 of d-limonene is greater than 2000 mg/kg bw in rabbits therefore it is not classified according to Directive 67/548/EEC and CLP Regulation (EC) No 1272/2008.
Executive summary:

In an acute dermal toxicity study (limit test), a group of 10 New Zealand White rabbits were administered a single dermal dose of d-limonene at 5000 mg/kg bw on clipped abraded abdominal skin using an occlusive patch for 24 hours. Animals were then observed for mortality, clinical signs, bodyweights and dermal reactions for 7 days and were all macroscopically necropsied after sacrifice.

 

No deaths occurred throughout the study. Normal body weight gain was observed in all animals. At necropsy, macroscopic examination of main organs showed no abnormalities. Adverse dermal reactions noted were slight to moderate erythema and edema, which completely recovered to normal within five days. The acute dermal LD50 was found to be greater than 5000 mg/kg bw.

 

The acute dermal LD50 of d-limonene is greater than 2000 mg/kg bw in rabbits therefore it is not classified according to Directive 67/548/EEC and CLP Regulation (EC) No 1272/2008.

Endpoint:
acute toxicity: dermal
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
L-Limonene is the S-isomer of d-limonene. Therefore, data on l-limonene can be used for extrapolation to d-limonene. See read-across justification document in section 13.
Reason / purpose:
read-across source
Preliminary study:
Not applicable
Key result
Sex:
not specified
Dose descriptor:
LD50
Effect level:
> 5 000 mg/kg bw
Based on:
test mat.
Mortality:
No mortality was observed.
Clinical signs:
No
Body weight:
No data
Gross pathology:
No data
Other findings:
- Dermal reactions: moderate redness (3/10 rabbits) and moderate edema (6/10 rabbits)

None

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The dermal LD50 for l-limonene is higher than 5000 mg/kg bw in rabbits therefore it is not classified according to Directive 67/548/EEC and CLP Regulation (EC) No 1272/2008.
Executive summary:

In an acute dermal toxicity study, 10 rabbits were administered a single dermal dose of l-limonene at 5000 mg/kg bw. Animals were then observed for mortality and clinical signs of toxicity for 14 days. No deaths and clinical signs of toxicity occurred during the observation period. Dermal reactions noted were moderate redness (3/10 rabbits) and moderate edema (6/10 rabbits) at the site of application.

 

The dermal LD50 for l-limonene is higher than 5000 mg/kg bw in rabbits therefore it is not classified according to Directive 67/548/EEC and CLP Regulation (EC) No 1272/2008.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
5 000 mg/kg bw
Quality of whole database:
One acute dermal toxicity study was available for several monocyclic terpene substances, i.e. d-limonene, l-limonene, terpinolene and gamma-terpinene all showing LD50 values >5000 mg/kg bw. Although these studies were old and briefly described, they all show consistent results about all these structure-related substances. Morevover, the low dermal toxicity is consistent with and confirmed by the low oral toxicity of tested monocyclic terpene substances.

Additional information

The key study selected for acute oral toxicity endpoint is a study performed with the read-across substance dipentene multi-constituent, according to OECD Guideline 423. The oral LD50 was found to be greater than 2000 mg/kg bw in rats. One death was observed in the read-across study at 2000 mg/kg bw. However, four additional studies, old and briefly described, were performed with d-limonene or dipentene. All these studies demonstrated oral LD50 greater than 4400 mg/kg bw in rats or mice. Therefore the LD50 of d-limonene is most probably higher than 5000 mg/kg bw in rats. One acute dermal toxicity study was available for several monocyclic terpene substances, i.e. d-limonene, l-limonene, terpinolene and gamma-terpinene all showing LD50 values > 5000 mg/kg bw. Although these studies were old and briefly described, they all show consistent results about all these structure-related substances. Moreover, the low dermal toxicity is consistent with and confirmed by the low oral toxicity of tested monocyclic terpene substances.

For further information on read-across justification, see section 13: point "read-across approach"


Justification for selection of acute toxicity – oral endpoint
The key study is the most recent study conducted according to GLP on a read-across substance

Justification for selection of acute toxicity – inhalation endpoint
No study was available and it was not necessary to provide one because acute toxicity is already assessed via two different routes of exposure. Acute toxicity studies by oral and dermal routes showed very low toxicity, with high LD50 values.

Justification for selection of acute toxicity – dermal endpoint
No robust study summary was chosen for this endpoint because a weight of evidence approach was adopted; therefore, it was not possible to select only one of the studies used for this endpoint.

Justification for classification or non-classification

Oral and dermal LD50 are potentially higher than 5000 mg/kg bw in rats and rabbits, respectively, therefore d-limonene does not need to be classified according to CLP Regulation (EC) No 1272/2008 and GHS.