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Description of key information

In a GLP study conducted on alpha-pinene multiconstituent according to the OECD 423 guideline the oral LD50 cut-off value (rats) was 500 mg/kg bw.

In a GLP study conducted on alpha-pinene multiconstituent according to the OECD 402 guideline the dermal LD50 (rats) was higher than 2000 mg/kg bw

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records
Reference
Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May-June 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
read-across: supporting information
Qualifier:
according to
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.1 (Acute Toxicity (Oral))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
23 October 2015
Test type:
standard acute method
Limit test:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS:
Sprague Dawley rats supplied by Elevage JANVIER LABS (53940 Le Genest St Isle – France)
Age at study initiate: 8 or 9 weeks old
Weight at study initiation: 185-230g
Housing: Animals were housed by group of three in solid bottomed clear polycarbonate cages with stainless steel mesh lid.
Fasting period before study: Food was removed on Day 1 and then redistributed 4 h after administration of the test item.
Diet: Foodstuff (SAFE, A04), ad libitum
Water: Drinking water (tap water from public distribution system), ad libitum
Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS:
Air recycling: at least 10 cycles per hour,
Temperature: 22° C ± 3° C,
Relative humidity: from 30 % to 70 %,
Lighting: circadian cycle (12 hrs day / 12 hrs darkness).
Route of administration:
oral: gavage
Vehicle:
other: for 2000 mg/kg dose no vehicle, for 300 mg/kg vehicle is olive oil
Details on oral exposure:
In the first step of the study, the test item was administered by gavage under a volume of 2.33 mL/kg body weight (corresponding to 2000 mg/kg, according to the calculated relative density).

In the second and the third step of the study, 0.35 mL of the test item (corresponding to 300 mg/kg) was added to 1.98 mL of olive oil.
Doses:
2000 mg/kg and 300 mg/kg
No. of animals per sex per dose:
3 female rats for 2000 mg/kg
6 female rats at 300 mg/kg
Control animals:
other: historical data: Study No. TAO423-2016-002
Details on study design:
Clinical observations and mortality were recorded every day for 14 days
Animals were weighed on day D0 (just before administering the test item) then on D2, D7, and D14.
On D14, the animals were anaesthetised with sodium pentobarbital and administration continued to fatal levels. Macroscopic observations were entered on individual autopsy sheets.
Only those organs likely to be modified in cases of acute toxicity were examined. No organ was removed and preserved in view to microscopic examinations.
Statistics:
none
Key result
Sex:
female
Dose descriptor:
LD50 cut-off
Effect level:
> 500 mg/kg bw
Based on:
test mat.
Mortality:
Three mortalities were noted in animals treated at the dose of 2000 mg/kg body weight, at 24 hours post-dose.

No mortality was noted in the animals treated at the dose of 300 mg/kg body weight.
Clinical signs:
At the dose of 2000 mg/kg the mortalities were preceded by a decrease in spontaneous activity (1/3) at 30 minutes post-dose.
At the dose of 300 mg/kg no clinical signs related to the administration of the test item were observed during the study.
Body weight:
The body weight evolution of the animals remained normal throughout the study at the dose of 300 mg/kg.
No data are available at the dose of 2000 mg/kg (animal found animals found dead at T0 + 21 hours and 19 minutes).
Gross pathology:
At the dose of 200 mg/kg the macroscopic examination of the animals revealed a thinning of the corpus (2/3).
At the dose of 300 mg/kg the macroscopic examination of the animals at the end of the study did not reveal treatment related changes.
Interpretation of results:
Category 4 based on GHS criteria
Conclusions:
The LD50 of test item alpha-pinene multiconstituent is higher than 300 mg/kg body weight and lower than 2000 mg/kg by oral route in the rat.
In accordance with guideline OECD 423, the LD50 cut-off of the test item may be considered as 500 mg/kg body weight by oral route in the rat.

According to the criteria for classification, packaging and labelling of dangerous substances and preparations in accordance with Regulation EC No. 1272/2008, test item alpha-pinene multiconstituent has to be classified in category 4. The signal word “Warning” and hazard statement H302 “Harmful if swallowed” are required
Executive summary:

The test item alpha-pinene multiconstituent was administered to a group of 3 female Sprague Dawley rats at the dose of 2000 mg/kg body weight and then to a group of 6 female Sprague Dawley rats at the dose of 300 mg/kg body weight. The experimental protocol was established according to the official method as defined in the O.E.C.D. Test Guideline No. 423 dated December 17th, 2001 and the test method B.1tris of the Council regulation No. 440/2008.

Three mortalities were noted in animals treated at the dose of 2000 mg/kg body weight, at 24 hours post-dose.

The mortalities were preceded by a decrease in spontaneous activity (1/3) at 30 minutes post-dose. No other signs of systemic toxicity were noted.

Rigor mortis were noted before the necropsy in two animals (2/3). The macroscopic examination of the animals revealed a thinningof the corpus (2/3).

 

No mortality was noted in the animals treated at the dose of 300 mg/kg body weight.

No clinical signs related to the administration of the test item were observed during the study.

The body weight evolution of the animals remained normal throughout the study.

The macroscopic examination of the animals at the end of the study did not reveal treatment related changes.

In conclusion, the LD50 of test item alpha-pinene multiconstituent is higher than 300 mg/kg body weight and lower than 2000 mg/kg by oral route in the rat.

In accordance with guideline OECD 423, the LD50 cut-off of the test item may be considered as 500 mg/kg body weight by oral route in the rat.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LD50
500 mg/kg bw
Quality of whole database:
GLP study following OECD guideline 423

Acute toxicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Acute toxicity: via dermal route

Link to relevant study records
Reference
Endpoint:
acute toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
read-across: supporting information
Qualifier:
according to
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.3 (Acute Toxicity (Dermal))
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Test type:
standard acute method
Limit test:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Elevage Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: 7 weeks old (male) and 8 weeks old (female)
- Weight at study initiation: from 253g to 286 g (male) and from 195g to 217g (female)
- Housing: Animals were housed in solid-bottomed clear polycarbonate cages with a stainless steel mesh lid; During the treatment, the animals were kept in individual cages and after the removal of the patch on Day 1, the animals were put into their cage by group of 5.
- Diet: Foodstuff (SAFE, A04), ad libitum
- Water: Drinking water (tap water from public distribution system), ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25 °C
- Humidity: 30-70 %
- Air changes: At least 10 changes/h
- Photoperiod: 12 h dark / 12 h light
Type of coverage:
semiocclusive
Vehicle:
unchanged (no vehicle)
Details on dermal exposure:
Approximately 24 h before the treatment, fur was removed from the dorsal area of the trunk of the test animals by clipping.

TEST SITE
- Area of exposure: Dorsal area of the trunk
- % coverage: Approximately 10 % of the body surface area
- Type of wrap if used: Test material was applied by topical application, under porous gauze dressing.

REMOVAL OF TEST SUBSTANCE
- Time after start of exposure: 24 h

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2000 mg/kg bw
- Dose volume: mL/kg bw (corresponding to 2000 mg/kg bw according to the density of the test item)
- Constant volume or concentration used: Yes
Duration of exposure:
24 h
Doses:
2000 mg/kg bw
No. of animals per sex per dose:
5
Control animals:
not specified
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing:
Clinical signs and mortality: Animals were observed for any behavioural or toxic effects on the major physiological functions at 1, 3 and 5 h 24 hours and 48 hours after administration of the test item and continued on the following days if necessary. Clinical observations and mortality were recorded every day for 14 days.
- The integrity of the skin on the application site was noted.
Bodyweight was recorded on Days 0 (just before administering test item), 2, 7 and 14.
- Necropsy of survivors performed: Yes; On Day 14, the animals were anaesthetised with sodium pentobarbital and administration continued to fatal levels and macroscopic examination was performed.
Statistics:
None
Preliminary study:
Not applicable
Key result
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
No mortality was observed.
Clinical signs:
No systemic clinical signs related to the administration of the test item were observed.
Cutaneous reactions (erythema and dryness) were noted in all animals (10/10) at 24 h post-dose and were totally reversible on day 13.
Body weight:
Body weight evolution of the animals remained normal throughout the study compared to the historical control group.
Gross pathology:
No macroscopic abnormalities were observed at necropsy.
Other findings:
None

None

Interpretation of results:
GHS criteria not met
Conclusions:
The acute dermal LD50 of alpha-pinene multiconstituent is higher than 2000 mg/kg bw in rats therefore it is not classified according to CLP Regulation (EC) n° 1272/2008 and UN GHS.
Executive summary:

In an acute dermal toxicity study performed according to OECD Guideline 402 and in compliance with GLP, a group of Sprague Dawley rats (5/sex/dose) was given a single dermal application of alpha-pinene multiconstituent at 2000 mg/kg bw. The test item was applied on dorsal area of the trunk representing approximately 10% of the total body surface area of the animals. The test site was then covered by a semi-occlusive dressing for 24 h. Animals were then observed for mortality, clinical signs and bodyweights for 14 days and were all sacrificed for macroscopic examination.

No mortality occurred during the study. No systemic clinical signs related to the administration of the test item were observed. Cutaneous reactions (erythema and dryness) were noted in all animals (10/10) at 24-hour post-dose and were totally reversible on day 13. Body weight gain of the animals remained normal throughout the study. The macroscopic examination of the animals at the end of the study did not reveal any treatment-related changes.

In conclusion, the LD50 of test item alpha-pinene multiconstituent is higher than 2000 mg/kg body weight by dermal route in the rat. Test item alpha-pinene multiconstituent does not have to be classified in accordance with Regulation EC No. 1272/2008 on classification, labelling and packaging of substances and mixtures and UN GHS.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
2 000 mg/kg bw
Quality of whole database:
GLP study following OECD guideline 402

Additional information

A GLP study conducted according to OECD guideline 423 was performed in rats with alpha-pinene multiconstituent. Two doses, 2000 and 300 mg/kg bw, were tested. Three mortalities were noted in animals treated at the dose of 2000 mg/kg body weight, at 24 hours post-dose. No mortality was noted in the animals treated at the dose of 300 mg/kg body weight. The LD50 of test item alpha-pinene multiconstituent is higher than 300 mg/kg body weight and lower than 2000 mg/kg by oral route in the rat. In accordance with OECD guideline 423, the LD50 cut-off of the test item may be considered as 500 mg/kg bw by oral route in rats.

In a GLP study conducted according to OECD guideline 402, alpha-pinene multiconstituent was administered to ten Sprague Dawley rats (5 males and 5 females) at the single dose of 2000 mg/kg body weight. No mortality occured during the study and no systemic clinical signs related to the administration of the test item were observed. The macroscopic examination of the animals at the end of the study did not reveal any treatment-related changes. The LD50 of test item alpha-pinene multiconstituent is higher than 2000 mg/kg bw.

Justification for classification or non-classification

Considering an oral LD50 of 500 mg/kg and according to the criteria of Regulation EC No. 1272/2008, (-)-alpha-pinene has to be classified in category 4 for acute oral toxicity. The signal word “Warning” and hazard statement H302 “Harmful if swallowed” are required.

As LD50 for acute dermal toxicity is higher than 2000 mg/kg bw, the test item is not classified according to CLP Regulation (EC) n° 1272/2008.