Registration Dossier

Administrative data

Description of key information

Acute oral toxicity: Key study. Test method according to OECD 423, GLP study. The LD50 of the test item is higher than 2000 mg/kg body weight by oral route in the rat. The LD50 cut-off may be considered to be 5000 mg/kg body weight by oral route in the rat.

Acute inhalation toxicity: Data waiving (study scientifically not necessary): According to REACH Annex VIII, column 2: In addition to the oral route, for substances other than gases, the information mentioned under 8.5.2 to 8.5.3 shall be provided for at least one other route. The information is provided for dermal route.

Acute dermal toxicity: Key study. The LD50 of the test item is higher than 2000 mg/kg body weight by dermal route in rats.

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:
experimental study
Adequacy of study:
key study
Study period:
09 May 2018 - 29 May 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test type:
acute toxic class method
Limit test:
yes
Species:
rat
Strain:
Sprague-Dawley
Remarks:
(SPF Caw)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Elevage JANVIER LABS (53940 Le Genest St Isle – France)
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 8 or 9 weeks
- Weight at study initiation: 212.3 g (SD = 17.4 g)
- Fasting period before study: Food was removed on D-1 and then redistributed 4 hours after the test item administration.
- Housing: Rats were housed by group of three in solid-bottomed clear polycarbonate cages with a stainless steel mesh lid. Each cage contains sawdust bedding which was changed at least 2 times a week. Each cage was installed in conventional air conditioned animal husbandry.
- Diet (e.g. ad libitum): Ad libitum. Teklad Global 16% Protein Rodent Diet (ENVIGO 2016).
- Water (e.g. ad libitum): Ad libitum. Drinking water (tap-water from public distribution system). Microbiological and chemical analyses of the water were carried out once every six months by Bureau Veritas – Eurofins (FRANCE).
- Acclimation period: the animals were acclimatized for at least 5 days before treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19ºC to 25ºC
- Humidity (%): 30 to 70%
- Air changes (per hr): >10 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light (07.00 to 19.00) and 12 hours dark cycle.
Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
MAXIMUM DOSE VOLUME APPLIED: 2.09 mL/kg body weight (corresponding to 2 g/kg, according to the calculated density)

CLASS METHOD (if applicable)
- Rationale for the selection of the starting dose: Available information suggests that mortality is unlikely at the highest starting dose level (2000 mg/kg body weight). Hence, the study is initiated with the starting dose of 2000 mg/kg body weight.
Doses:
2000 mg/kg bw
No. of animals per sex per dose:
6 (3 female rats per step)
Control animals:
yes
Remarks:
(study performed on three animals receiving distilled water under requirements of OECD Guideline 423)
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: At each step, the animals were observed four times on test day 0 (day of administration), i.e. at T0+30 min, T0+1h, T0+3h and T0+4h, and once daily during days 1 to 14 post administration. The body weights were recorded on test day 0 (just before administration) then on D2, D7, and D14.
- Necropsy of survivors performed: yes. At termination, gross pathological findings were recorded and reported.
- Other examinations performed:
Clinical observations included: spontaneous activity, Preyer’s reflex (noise), respiratory rate, convulsions, tremors, body temperature, muscle tone, palpebral opening, pupil appearance, salivation, lachrymation, righting reflex, back hair appearance.
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Key result
Sex:
female
Dose descriptor:
LD50 cut-off
Effect level:
5 000 mg/kg bw
Based on:
test mat.
Mortality:
No mortality occurred during the study.
Clinical signs:
A decrease of spontaneous activity (2/6) was noted at 30 minutes post-dose. The animals recovered a normal activity at 1 hour post-dose.
No other clinical signs were observed.
Body weight:
The body weight evolution of the animals remained normal during the study.
Gross pathology:
The macroscopic examination of the animals at the end of the study did not reveal treatment related changes.

Table 1: Body weight and weight gain in grams

Females

D0

D2

D2-D0

D7

D7-D0

D14

D14-D0

Rf 2500

193

220

27

250

57

263

70

Rf 2501

196

210

14

235

39

248

52

Rf 2502

204

201

-3

241

37

267

63

Rf 2544

232

232

0

265

33

285

53

Rf 2545

232

230

-2

278

46

296

64

Rf 2546

217

226

9

260

43

280

63

MEAN

212.3

219.8

7.5

254.8

42.5

273.2

60.8

SD

17.4

12.2

11.7

16.0

8.4

17.2

7.0

Interpretation of results:
other: Not classified (CLP Regulation EC no. 1272/2008)
Conclusions:
The LD50 of the test item is higher than 2000 mg/kg body weight by oral route in the rat. The LD50 cut-off may be considered to be 5000 mg/kg body weight by oral route in the rat.
Executive summary:

The acute oral toxicity of the test item has been tested in accordance with OECD Test Guideline 423, following GLP. 6 female Sprague-Dawley rats divided in 2 groups were administered sequentially with test item by oral gavage. The first set of 3 female rats was given a single dose of 2000 mg/kg body weight. No mortality was observed at this dose level, so the second set of 3 female rats was treated at the same dose level. No mortality was observed at this dose level either. The body weight evolution of the animals remained normal during the study. A decrease of spontaneous activity (2/6) was noted at 30 minutes post-dose. The animals recovered a normal activity at 1 hour post-dose. No other clinical signs were observed. The macroscopic examination of the animals at the end of the study did not reveal treatment related effects. The LD50 of the test item is higher than 2000 mg/kg body weight by oral route in the rat. As per OECD Test Guideline 423, the LD50 cut-off of the test item may be considered to be 5000 mg/kg body weight by oral route in the rat.

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Principles of method if other than guideline:
No data on test method
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
not specified
Sex:
not specified
Route of administration:
oral: unspecified
Vehicle:
not specified
Doses:
No data
No. of animals per sex per dose:
No data
Control animals:
not specified
Sex:
not specified
Dose descriptor:
LD50
Effect level:
> 5 000 mg/kg bw

The acute oral LD50 value in rats was reported as greater than 5 g/kg bw.

Interpretation of results:
other: Not classified (CLP Regulation EC no. 1272/2008)
Conclusions:
The acute oral LD50 value in rats was reported as greater than 5 g/kg bw.
Executive summary:

The acute oral LD50 value in rats was reported as greater than 5 g/kg bw.

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Principles of method if other than guideline:
No data on test method
GLP compliance:
no
Species:
rat
Strain:
not specified
Sex:
not specified
Route of administration:
oral: unspecified
Vehicle:
not specified
Control animals:
not specified
Sex:
not specified
Dose descriptor:
LD50
Effect level:
> 5 000 mg/kg bw
Based on:
test mat.
Interpretation of results:
other: Not classified (CLP Regulation EC no. 1272/2008)
Conclusions:
The acute oral LD50 value of the test substance is greater than 5000 mg/kg bw in rats.
Executive summary:

According to a peer reviewed article, the acute oral LD50 value of the test substance is greater than 5000 mg/kg bw in rats.

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Principles of method if other than guideline:
No data on test method
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
not specified
Sex:
not specified
Route of administration:
oral: unspecified
Vehicle:
not specified
Doses:
No data
No. of animals per sex per dose:
No data
Control animals:
not specified
Sex:
not specified
Dose descriptor:
LD50
Effect level:
1 680 mg/kg bw
95% CL:
> 1 460 - < 1 900

The acute oral LD50 value in rats was reported as 1.68 g/kg bw.

Interpretation of results:
study cannot be used for classification
Conclusions:
The acute oral LD50 value in rats was reported as 1.68 g/kg bw.
Executive summary:

The acute oral LD50 value in rats was reported as 1.68 g/kg bw (1.46 - 1.90 g/kg).

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Principles of method if other than guideline:
No data on test method
GLP compliance:
no
Species:
rat
Strain:
not specified
Sex:
not specified
Route of administration:
oral: unspecified
Vehicle:
not specified
Control animals:
not specified
Sex:
not specified
Dose descriptor:
LD50
Effect level:
4.39 mL/kg bw
Based on:
test mat.
Interpretation of results:
other: Not classified (CLP Regulation EC no. 1272/2008)
Conclusions:
The acute oral LD50 value of the test substance is 4.39 mL/kg bw in rats (equivalent to 3789 mg/kg bw).
Executive summary:

According to a peer reviewed article, the acute oral LD50 value of the test substance is 4.39 mL/kg bw in rats (equivalent to 3789 mg/kg bw).

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Principles of method if other than guideline:
No data on test method
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
not specified
Sex:
not specified
Route of administration:
oral: unspecified
Vehicle:
not specified
Doses:
No data
No. of animals per sex per dose:
No data
Control animals:
not specified
Sex:
not specified
Dose descriptor:
LD50
Effect level:
3 700 mg/kg bw
Remarks on result:
other: confidence limit: 2300-5100 mg/kg bw

The acute oral LD50 value in rats was reported as 3.7 g/kg bw (2.3 -5.1 g/kg bw)

Interpretation of results:
other: Not classified (CLP Regulation EC no. 1272/2008)
Conclusions:
The acute oral LD50 value in rats was reported as 3.7 g/kg bw (2.3 -5.1 g/kg bw)
Executive summary:

The acute oral LD50 value in rats was reported as 3.7 g/kg bw (2.3 -5.1 g/kg bw)

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Principles of method if other than guideline:
No data on test method
GLP compliance:
no
Species:
rat
Strain:
not specified
Sex:
not specified
Route of administration:
oral: unspecified
Vehicle:
not specified
Control animals:
not specified
Sex:
not specified
Dose descriptor:
LD50
Effect level:
> 5 000 mg/kg bw
Based on:
test mat.
Interpretation of results:
other: Not classified (CLP Regulation EC no. 1272/2008)
Conclusions:
The acute oral LD50 value of the test substance is greater than 5000 mg/kg bw in rats.
Executive summary:

According to a peer reviewed article, the acute oral LD50 value of the test substance is greater than 5000 mg/kg bw in rats.

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Principles of method if other than guideline:
No data on test method.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
not specified
Sex:
not specified
Route of administration:
oral: unspecified
Vehicle:
not specified
Doses:
No data
No. of animals per sex per dose:
No data
Control animals:
not specified
Sex:
not specified
Dose descriptor:
LD50
Effect level:
> 5 000 mg/kg bw

The acute oral LD50 value in rats was reported as greater than 5 g/kg bw.

Interpretation of results:
other: Not classified (CLP Regulation EC no. 1272/2008)
Conclusions:
The acute oral LD50 value in rats was reported as greater than 5 g/kg bw.
Executive summary:

The acute oral LD50 value in rats was reported as greater than 5 g/kg bw.

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:
documentation insufficient for assessment
Remarks:
(Original reference in Japanese language)
Principles of method if other than guideline:
- Principle of test: Standard acute method
GLP compliance:
no
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:
other: Not classified (CLP Regulation EC no. 1272/2008)
Conclusions:
The oral LD50 for d-limonene is higher than 5000 mg/kg bw in mice therefore it is not classified according to the CLP Regulation (EC) N° (1272-2008).
Executive summary:

In an acute oral toxicity study, groups (10/sex) of ddN 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 the CLP Regulation (EC) N° (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:
documentation insufficient for assessment
Remarks:
(Original reference in Japanese language)
Principles of method if other than guideline:
- Principle of test: Standard acute method
GLP compliance:
no
Test type:
standard acute method
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
(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:
other: Not classified (CLP Regulation EC no. 1272/2008)
Conclusions:
The oral LD50 for d-limonene is higher than 2000 mg/kg bw in rats therefore it is not classified according to the CLP Regulation (EC) N° (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 the CLP Regulation (EC) N° (1272-2008).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
Value:
5 000 mg/kg bw
Quality of whole database:
Key study with Klimisch score = 1

Acute toxicity: via inhalation route

Link to relevant study records

Referenceopen allclose all

Endpoint:
acute toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
According to REACH Annex VIII, column 2: In addition to the oral route (Annex VII, 8.5.1.), for substances other than gases, the information mentioned under 8.5.2 to 8.5.3 shall be provided for at least one other route. The information is provided for dermal route.
Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
Test method was not according to any guideline. The species used was the rabbit. However, the recommended species for the acute inhalation toxicity is the rat. No GLP.
Principles of method if other than guideline:
- Principle of test: Studies on the pharmacological expectorant activity of nutmeg oil and camphene given by inhalation to rabbits arranged for the collection of respiratory tract fluid.
- Short description of test conditions: see below
- Parameters analysed / observed: volumen of respiratory tract fluid, expectorant activity and odor
GLP compliance:
no
Test type:
other: Study of the pharmacological expectorant activity of test substance
Limit test:
no
Species:
rabbit
Strain:
not specified
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: Young
- Weight at study initiation: 2-3 kg
- Housing: They were housed in boarding cages, one animal per cage.
- Diet (e.g. ad libitum): They were fed a standard rabbit ration of mixed natural foods, salts, and vitamins.
Route of administration:
inhalation: vapour
Type of inhalation exposure:
nose only
Vehicle:
other: ethanol
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus:
In preparation for the collection of respiratory tract fluid, the rabbits were anesthetized with urethane (ethyl carbamate) given by intraperitoneal injection in a dose of 4.0 to 5.0 mL/kg body weight of a 25% (w/v) solution in distilled water. This produced a light anesthesia lasting over 24 hours with no reported effect on the volume and composition of respiratory tract fluid except that it augmented the concentration of potassium ion.
One arm of a T-shaped cannula was ligated into the trachea, a collecting tube attached to the second arm, and the third arm was connected with a reservoir of conditioned air maintained at 39 ºC and saturated with water vapor. The conditioned air was prepared by mixing laboratory air with steam from a thermostatically controlled water vaporizer. Steam generated by this vaporizer volatilized the test item which was carried to the conditioned air reservoir from which it was inhaled by each anesthetized rabbit.
Respiratory tract fluid was collected for a control period of two to four hours. The collecting tube was then replaced by a cleaned, empty tube, test item added to the vaporizer, and respiratory tract fluid collected for a subsequent period of four to six hours.


Analytical verification of test atmosphere concentrations:
not specified
Duration of exposure:
>= 4 - <= 6 h
Concentrations:
1, 3, 9, 27, 81, and 243 mg/kg
No. of animals per sex per dose:
4-6 male rabbits per dose
Control animals:
other: The controls were the same animals, before being treated.
Details on study design:
- Duration of observation period following administration: 24 hours, which is a standard method of expressing the volume output of respiratory tract fluid.
Key result
Sex:
male
Dose descriptor:
LC0
Effect level:
>= 243 other: mg/kg
Based on:
test mat.
Exp. duration:
6 h
Mortality:
No animals died during the study.
Clinical signs:
other: Inhalation of camphene by urethanized rabbits in doses 3-27 mg/kg produced significant increase in volume output of respiratory tract fluid, accompanied by decrease in specific gravity and increase in total solids. Dose-dependent increase in volume output

The LC 0 was equal or greater than 243 mg/kg.

Interpretation of results:
study cannot be used for classification
Conclusions:
The LC 0 was equal or greater than 243 mg/kg.
Executive summary:

Camphene was given by inhalation to male rabbits that were anesthetized with urethane (ethyl carbamate) for 24 hours and were arranged for collection of respiratory tract fluid. Test substance was dissolved in 1 mL of ethanol in amounts of 1, 3, 9, 27, 81, and 243 mg. Each dose was given to 4 to 6 rabbits. Respiratory tract fluid was collected for a control period of two to four hours. The collecting tube was then replaced by a cleaned, empty tube, test item added to the vaporizer, and respiratory tract fluid collected for a subsequent period of four to six hours. Steam generated by the water vaporizer volatilized the test item which was carried to the conditioned air reservoir from which it was inhaled by each anesthetized rabbit. Inhalation of camphene by urethanized rabbits in doses 3-27 mg/kg produced significant increase in volume output of respiratory tract fluid, accompanied by decrease in specific gravity and increase in total solids. Dose-dependent increase in volume output of respiratory tract fluid varied with the season. The expectorant action began to disappear following inhalation of a dose of 81 mg/kg, which is well below the level detectable by odour (243 mg/kg) and far below the probably fatal dose. Based on these results, the LC0 was established to be equal or greater than 243 mg/kg.

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Principles of method if other than guideline:
No data on test method
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
not specified
Sex:
not specified
Route of administration:
inhalation: vapour
Type of inhalation exposure:
not specified
Vehicle:
not specified
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Test animals inhaled saturated vapours of alpha pinene from steam distillation of wood.


Analytical verification of test atmosphere concentrations:
not specified
Concentrations:
26 mg/L
No. of animals per sex per dose:
no data
Control animals:
not specified
Key result
Sex:
not specified
Dose descriptor:
LC50
Effect level:
625 other: mg/L/min
Based on:
test mat.
Remarks on result:
other: Effect level is the LC for the shortest period causing death
Interpretation of results:
study cannot be used for classification
Conclusions:
The LC50 of the test item (for the shortest period causing death) is 625 mg/l/min in rats.
Executive summary:

Alpha pinene was given to rats through inhalation of saturated vapours (26 mg/L of alpha pinene) obtained from steam distillation of wood. The LC50 of the test item (for the shortest period causing death) was found to be 625 mg/l/min.

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:
key study
Study period:
09 May 2018 - 29 May 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test type:
fixed dose procedure
Limit test:
yes
Species:
rat
Strain:
Sprague-Dawley
Remarks:
(SPF Caw)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Elevage JANVIER LABS (53940 Le Genest St Isle – France)
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 8 or 9 weeks
- Weight at study initiation: 220.7 g (SD = 20.8 g)
- Fasting period before study: not specified.
- Housing: During the treatment, the animals were kept in individual cages. On D1, the animals were put together into their cage. The rats were kept in solid-bottomed clear polycarbonate cages with a stainless steel mesh lid. Each cage contains dust free wood shavings which were changed at least 2 times a week. Each cage was installed in conventional air conditioned animal husbandry.
- Diet (e.g. ad libitum): Ad libitum. Teklad Global 16% Protein Rodent Diet (ENVIGO 2016).
- Water (e.g. ad libitum): Ad libitum. Drinking water (tap-water from public distribution system). Microbiological and chemical analyses of the water were carried out once every six months by Bureau Veritas – Eurofins (FRANCE).
- Acclimation period: the animals were acclimatized for at least 5 days before treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19ºC to 25ºC
- Humidity (%): 30 to 70%
- Air changes (per hr): >10 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light (07.00 to 19.00) and 12 hours dark cycle.
Type of coverage:
semiocclusive
Vehicle:
unchanged (no vehicle)
Details on dermal exposure:
TEST SITE
- Area of exposure: dorsal area of the trunk
- % coverage: at least 10% of the body surface area.
- Type of wrap if used: porous gauze dressing (50 mm x 50 mm non-woven swab of 4-layer patch from MEDISTOCK) secured in position with a strip of surgical adhesive tape (50 mm wides hypoallergenic microporeTM adhesive tape from 3M).

REMOVAL OF TEST SUBSTANCE
- Washing (if done): no
- Time after start of exposure: 24h

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2000 mg/kg body weight
- Concentration (if solution): N/A
- Constant volume or concentration used: 2.09 mL/kg body weight
- For solids, paste formed: N/A
Duration of exposure:
24 h
Doses:
Range finding study: 2000 mg/kg body weight
Main study: 2000 mg/kg body weight
No. of animals per sex per dose:
Range finding study: 1 female per dose
Main study: 2 females per dose
Control animals:
yes
Remarks:
(study performed on three females receiving distilled water by topical application under requirements of OECD Guideline 402)
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: At each step, the animals were observed three times on test day 0 (day of administration), i.e. at T0+30 min, T0+3h and T0+5h, and once daily during days 1 to 14 post administration. The body weights were recorded on test day 0 (just before administration) then on D2, D7, and D14.
- Necropsy of survivors performed: yes. At termination, gross pathological findings were recorded and reported.
- Other examinations performed:
Clinical observations: spontaneous activity, Preyer’s reflex (noise), respiratory rate, convulsions, tremors, body temperature, muscle tone, palpebral opening, pupil appearance, salivation, lachrymation, righting reflex and treatment site (erythema, dryness of the skin, scab, etc.)
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
No mortality occurred during the study.
Clinical signs:
Erythema was noted in treated animals (2/3) at 48 hours post dose and was totally reversible on day 6.
Scab was noted in treated animals (2/3) between days 5 and 7.
Dryness of the skin was noted in treated animals (2/3) between days 8 and 10.
No other clinical signs were observed.
Body weight:
The body weight evolution of the animals remained normal throughout the study.
Gross pathology:
The macroscopic examination of the animal at the end of the study did not reveal treatment-related changes.

Table 1: Body weight and weight gain in grams

Females

D0

D2

D2-D0

D7

D7-D0

D14

D14-D0

Rf 2519

198

209

11

224

26

255

57

Rf 2576

239

248

9

250

11

266

27

Rf 2577

225

231

6

234

9

254

29

MEAN

220.7

229.3

8.7

236.0

15.3

258.3

37.7

SD

20.8

19.6

2.5

13.1

9.3

6.7

16.8

Interpretation of results:
other: No category (CLP Regulation EC no. 1272/2008)
Conclusions:
The LD50 of the test item is higher than 2000 mg/kg body weight by dermal route in rats.

Executive summary:

The acute dermal toxicity of the test item was studied according to OECD Guideline 402 (GLP study). 3 female Sprague-Dawley rats were tested for a exposure period of 24 h. Initially, a range finding study was conducted at a single dose of 2000 mg/kg bw in one animal. Based on the results of range finding two additional rats were tested at a dose of 2000 mg/kg bw. No mortality was observed. The body weight evolution of the animals remained normal during the study. Erythema was noted in treated animals (2/3) at 48 hours post dose and was totally reversible on day 6. Scab was noted in treated animals (2/3) between days 5 and 7. Dryness of the skin was noted in treated animals (2/3) between days 8 and 10. No other clinical signs were observed. The macroscopic examination of the animals at the end of the study did not reveal treatment related effects. Based on these results, the test item was found to be non toxic, with an LD50 > 2000 mg/kg bw.

Endpoint:
acute toxicity: dermal
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Principles of method if other than guideline:
No data on test method.
GLP compliance:
no
Limit test:
no
Species:
rabbit
Strain:
not specified
Sex:
not specified
Type of coverage:
not specified
Vehicle:
not specified
Duration of exposure:
No data
Doses:
No data
No. of animals per sex per dose:
No data
Control animals:
not specified
Sex:
not specified
Dose descriptor:
LD50
Effect level:
> 2 500 mg/kg bw

The acute dermal LD50 value in rabbits was reported as greater than 2.5 g/kg bw.

Interpretation of results:
other: Not classified (CLP Regulation EC no. 1272/2008)
Conclusions:
The acute dermal LD50 value in rabbits was reported as greater than 2.5 g/kg bw.
Executive summary:

The acute dermal LD50 value in rabbits was reported as greater than 2.5 g/kg bw.

Endpoint:
acute toxicity: dermal
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Principles of method if other than guideline:
No data on test method
GLP compliance:
no
Species:
rabbit
Strain:
not specified
Sex:
not specified
Type of coverage:
not specified
Vehicle:
not specified
Control animals:
not specified
Sex:
not specified
Dose descriptor:
LD50
Effect level:
> 5 000 mg/kg bw
Based on:
test mat.
Interpretation of results:
other: Not classified (CLP Regulation EC no. 1272/2008)
Conclusions:
The acute dermal LD50 value of the test substance was >5000 mg/kg bw in rabbits.
Executive summary:

According to a peer reviewed article, the acute dermal LD50 value of the test substance was >5000 mg/kg bw in rabbits.

Endpoint:
acute toxicity: dermal
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Principles of method if other than guideline:
No data on test method
GLP compliance:
no
Species:
rabbit
Strain:
not specified
Sex:
not specified
Type of coverage:
not specified
Vehicle:
not specified
Control animals:
not specified
Sex:
not specified
Dose descriptor:
LD50
Effect level:
> 5 000 mg/kg bw
Based on:
test mat.
Interpretation of results:
other: Not classified (CLP Regulation EC no. 1272/2008)
Conclusions:
The acute dermal LD50 value of the test substance was >5000 mg/kg bw in rabbits.
Executive summary:

According to a peer reviewed article, the acute dermal LD50 value of the test substance was >5000 mg/kg bw in rabbits.

Endpoint:
acute toxicity: dermal
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Principles of method if other than guideline:
No data on test method
GLP compliance:
no
Species:
rabbit
Strain:
not specified
Sex:
not specified
Type of coverage:
not specified
Vehicle:
not specified
Control animals:
not specified
Sex:
not specified
Dose descriptor:
LD50
Effect level:
> 5 000 mg/kg bw
Based on:
test mat.
Interpretation of results:
other: Not classified (CLP Regulation EC no. 1272/2008)
Conclusions:
The acute dermal LD50 value of the test substance was >5000 mg/kg bw in rabbits.
Executive summary:

According to a peer reviewed article, the acute dermal LD50 value of the test substance was >5000 mg/kg bw in rabbits.

Endpoint:
acute toxicity: dermal
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Principles of method if other than guideline:
No data on test method.
GLP compliance:
no
Limit test:
no
Species:
rabbit
Strain:
not specified
Sex:
not specified
Type of coverage:
not specified
Vehicle:
not specified
Duration of exposure:
No data
Doses:
No data
No. of animals per sex per dose:
No data
Control animals:
not specified
Key result
Sex:
not specified
Dose descriptor:
LD50
Effect level:
> 5 000 mg/kg bw

The acute dermal LD50 value in rabbits was reported as greater than 5 g/kg bw.

Interpretation of results:
other: Not classified (CLP Regulation EC no. 1272/2008)
Conclusions:
The acute dermal LD50 value in rabbits was reported as greater than 5 g/kg bw.
Executive summary:

The acute dermal LD50 value in rabbits was reported as greater than 5 g/kg bw.

Endpoint:
acute toxicity: dermal
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Principles of method if other than guideline:
No data on test method
GLP compliance:
no
Species:
rabbit
Strain:
not specified
Sex:
not specified
Type of coverage:
not specified
Vehicle:
not specified
Control animals:
not specified
Sex:
not specified
Dose descriptor:
LD50
Effect level:
> 5 000 mg/kg bw
Based on:
test mat.
Interpretation of results:
other: Not classified (CLP Regulation EC no. 1272/2008)
Conclusions:
The acute dermal LD50 value of the test substance was >5000 mg/kg bw in rabbits.
Executive summary:

According to a peer reviewed article, the acute dermal LD50 value of the test substance was >5000 mg/kg bw in rabbits.

Endpoint:
acute toxicity: dermal
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Principles of method if other than guideline:
No data on test method
GLP compliance:
not specified
Limit test:
no
Species:
rabbit
Strain:
not specified
Sex:
not specified
Type of coverage:
not specified
Vehicle:
not specified
Duration of exposure:
No data
Doses:
No data
No. of animals per sex per dose:
No data
Control animals:
not specified
Key result
Sex:
not specified
Dose descriptor:
LD50
Effect level:
> 2 500 mg/kg bw

The acute dermal LD50 value in rabbits was reported as greater than 2.5 g/kg bw.

Interpretation of results:
other: Not classified (CLP Regulation EC no. 1272/2008)
Conclusions:
The acute dermal LD50 value in rabbits was reported as greater than 2.5 g/kg bw.
Executive summary:

The acute dermal LD50 value in rabbits was reported as greater than 2.5 g/kg bw.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
Value:
2 000 mg/kg bw
Quality of whole database:
Key study with Klimisch score = 1

Additional information

Acute oral toxicity: Key study. The acute oral toxicity of the test item has been tested in accordance with OECD Test Guideline 423, following GLP. 6 femaleSprague-Dawleyrats divided in 2 groups were administered sequentially with test item by oral gavage.The first set of 3 female rats was given a single dose of 2000 mg/kg body weight. No mortality was observed at this dose level, so the second set of 3 female rats was treated at the same dose level. No mortality was observed at this dose level either. The body weight evolution of the animals remained normal during the study. A decrease of spontaneous activity (2/6) was noted at 30 minutes post-dose. The animals recovered a normal activity at 1 hour post-dose. No other clinical signs were observed.The macroscopic examination of the animals at the end of the study did not reveal treatment related effects. The LD50 of the test item is higher than 2000 mg/kg body weight by oral route in the rat. As per OECD Test Guideline 423, the LD50 cut-off of the test item may be considered to be 5000 mg/kg body weight by oral route in the rat.

Acute oral toxicity: Supporting studies. Individual acute toxicities of the main components are available from experimental tests.

Terpinolene: Peer reviewed publication (Opdyke DLJ, 1976). The acute oral LD50 value of the test substance is 4.39 mL/kg bw in rats (equivalent to 3789 mg/kg bw).

Camphene: Peer reviewed publication (Opdyke DLJ, 1975). The acute oral LD50 value in rats was reported as greater than 5 g/kg bw.

Camphene: Peer reviewed handbook (Patty's2001). The acute oral LD50 value in rats was reported as greater than 5 g/kg bw.

L-Limonene: Peer reviewed publication (Opdyke DLJ, 1978). The acute oral LD50 value of the test substance is greater than 5000 mg/kg bw in rats.

D-Limonene: Peer reviewed publication (Opdyke DLJ, 1975). The acute oral LD50 value of the test substance is greater than 5000 mg/kg bw in rats.

D-Limonene: Peer reviewed publication (Tsuji, 1975). The oral LD50 for d-limonene is higher than 2000 mg/kg bw in rats.

D-Limonene: Peer reviewed publication (Tsuji, 1975). The oral LD50 for d-limonene is higher than 5000 mg/kg bw in mice.

Alpha terpinene: Peer reviewed publication (Opdyke DLJ, 1976). The acute oral LD50 value in rats was reported as 1.68 g/kg bw.

Alpha pinene: Peer reviewed publication (Opdyke DLJ, 1978). The acute oral LD50 value in rats was reported as 3.7 g/kg bw.

Acute inhalation toxicity: Data waiving (study scientifically not necessary): According to REACH Annex VIII, column 2: In addition to the oral route, for substances other than gases, the information mentioned under 8.5.2 to 8.5.3 shall be provided for at least one other route. The information is provided for dermal route.

Acute inhalation toxicity: Supporting studies. Individual acute toxicities of the main components are available from experimental tests.

Camphene: Peer reviewed publication (Boyd EM, 1970). The LC 0 was equal or greater than 243 mg/kg.

Alpha pinene: Peer reviewed publication (Opdyke DLJ, 1978). The LC50 of the test item (for the shortest period causing death) is 625 mg/l/min in rats.

Acute dermal toxicity: Key study. The acute dermal toxicity of the test item was studied according to OECD Guideline 402 (GLP study). 3 female Sprague-Dawley rats were tested for a exposure period of 24 h. Initially, a range finding study was conducted at a single dose of 2000 mg/kg bw in one animal. Based on the results of range finding two additional rats were tested at a dose of 2000 mg/kg bw. No mortality was observed. The body weight evolution of the animals remained normal during the study.Erythema was noted in treated animals (2/3) at 48 hours post dose and was totally reversible on day 6. Scab was noted in treated animals (2/3) between days 5 and 7. Dryness of the skin was noted in treated animals (2/3) between days 8 and 10. No other clinical signs were observed.The macroscopic examination of the animals at the end of the study did not reveal treatment related effects.Based on these results, the test item was found to be non toxic, with an LD50 > 2000 mg/kg bw.

Acute dermal toxicity: Supporting studies. Individual acute toxicities of the main components are available from experimental tests.

Terpinolene: Peer reviewed publication (Opdyke DLJ, 1976). The acute dermal LD50 value of the test substance was >5000 mg/kg bw in rabbits.

Camphene: Peer reviewed publication (Opdyke DLJ, 1975). The acute dermal LD50 value in rabbits was reported as greater than 2.5 g/kg bw.

Camphene: Peer reviewed handbook (Patty's2001). The acute dermal LD50 value in rabbits was reported as greater than 2.5 g/kg bw.

L-Limonene: Peer reviewed publication (Opdyke DLJ, 1978). The acute dermal LD50 value of the test substance was >5000 mg/kg bw in rabbits.

D-Limonene: Peer reviewed publication (Opdyke DLJ, 1975). The acute dermal LD50 value of the test substance was >5000 mg/kg bw in rabbits.

Alpha pinene: Peer reviewed publication (Opdyke DLJ, 1978). The acute dermal LD50 value in rabbits was reported as greater than 5 g/kg bw.

Gamma terpinene: Peer reviewed publication (Opdyke DLJ, 1976). The acute dermal LD50 value of the test substance was >5000 mg/kg bw in rabbits.

Justification for classification or non-classification

Based on the available data, the substance is not classified for acute toxicity according to CLP Regulation (EC) no. 1272/2008.