Essential oil of Petitgrain obtained from the leaves and twigs of Citrus aurantium (Rutaceae) by distillation

Endpoint summary

Description of key information

Skin irritation (human, mouse, pig): not irritating

Eye irritation (OECD TG 437): no prediction on classification can be made

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1976

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

Pre testing: The material was pre-tested on twenty-five subects in order to determine wheater sodium lauryl sulfate pre-treatment was required. A patch of the materials was applied to normal sites on the volar forearms or backs for 48 hours under occlusion.
Maximization test: The material was applied under occlusion to the same site on volar forearm or back of all subects for five alternate-day 48 hour periods. The patch site was pre-treated for 24 hours with 2.5% aqueous sodium lauryl sulfate under occlusion. The challenge site was read on removal of the patch and 24 hours thereafter.

GLP compliance:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: RIFM#75-8-112 Petitgrain bigarde oil

Species:

other: human

Type of coverage:

occlusive

Vehicle:

other: petrolatum

Amount / concentration applied:

TEST MATERIAL
- Concentration: 8% in petrolatum

Duration of treatment / exposure:

48 hours

Observation period:

24 hours

Number of animals:

25 humans: 10 male and 15 female

Irritation parameter:

overall irritation score

Basis:

other: 25 humans 18+

Remarks:

Dose 8% in petrolatum

Time point:

48 h

Score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

overall irritation score

Basis:

other: 25 humans 18+

Remarks:

Dose 8% in petrolatum

Time point:

72 h

Score:

0

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

none

Other effects:

none

Interpretation of results:

other: not classified

Remarks:

based on CLP criteria (EC 1272/2008 and its updates)

Conclusions:

Under the conditions of this study, the testing material gave no instances of irritation. Based on this result, the test substance does not need to be classified for skin irritation in accordance with the criteria outlined in CLP (1272/2008/EC).

Executive summary:

The skin irritating potential of Petitgrain Bigarde Oil on humans was tested on 25 healthy adult volunteers. The material was applied under occlusion on the volar aspects of the forearm or backs for 48 hours. No subject had any irritation from the testing material. Under the conditions of this study, the testing material gave no instances of irritation. Based on this result, the test substance does not need to be classified for skin irritation in accordance with the criteria outlined in CLP (1272/2008/EC).

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1976

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

Pre-GLP, non guideline study

Principles of method if other than guideline:

- Principle of test: Irritration was evaluated at the non-irradiated sites in an associated phototoxicity study (conducted using mice)

GLP compliance:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: #75-112 Pettigrain Bigard Oil

Species:

mouse

Strain:

not specified

Details on test animals or test system and environmental conditions:

TEST ANIMALS
-Hairless mice

Amount / concentration applied:

dose 100%

Number of animals:

15

Irritation parameter:

other: phototoxicity

Basis:

animal: 1-15

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

None

Interpretation of results:

other: Not classified

Remarks:

Based on CLP criteria (EC 1272/2008 and its updates)

Conclusions:

Under the conditions of this test, Petitgrain Bigarde Oil is not an irritant to skin in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).

Executive summary:

In a dermal irritation study, irritation was evaluated at the non-irradiated sites in an associated phototoxicity study conducted on 15 hairless mice with neat Petitgrain Bigarde Oil. No irritation was observed. The substance did not show any irritant, skin staining or phototoxic effects. Under the conditions of this test, Petitgrain Bigarde Oil is not an irritant to skin in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).

Reference 3

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1976

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Principles of method if other than guideline:

- Principle of test: Irritration was evaluated at the non-irradiated sites in an associated phototoxicity study (conducted using miniature swine)

GLP compliance:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: #75-112 Pettigrain Bigard Oil

Species:

pig

Strain:

other: miniature swine

Amount / concentration applied:

100% dose

Number of animals:

5

Irritation parameter:

other: phototoxicity

Basis:

animal: 1-5

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

None

Interpretation of results:

other: Not classified

Remarks:

Based on CLP criteria (EC 1272/2008 and its updates)

Conclusions:

Under the conditions of this test, Petitgrain Bigarde Oil is not an irritant to skin in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).

Executive summary:

In a dermal irritation study, irritation was evaluated at the non-irradiated sites in an associated phototoxicity study conducted on 5 miniature swine with neat Petitgrain Bigarde Oil. No irritation was observed. The substance did not show any irritant, skin staining or phototoxic effects. Under the conditions of this test, Petitgrain Bigarde Oil is not an irritant to skin in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).

Reference 4

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1972

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Pre-GLP non OECD guideline study, with restrictions: purity/composition not specified

Principles of method if other than guideline:

- Principle of test: A primary irritation test of 19 kinds of natural and 43 kinds of synthetic perfumes was performed by the closed patch test on human skin in three ranges of concentrations.

GLP compliance:

no

Species:

other: human

Type of coverage:

other: Patch test

Preparation of test site:

not specified

Vehicle:

not specified

Controls:

no

Amount / concentration applied:

5%, 2%, 0.1%

Duration of treatment / exposure:

48 hours (5%), 24-72 hours (2%), 24-48 hours (0.1%)

Number of animals:

Human test group for testing material: dose 5%: 30 individuals, dose 2%: 30 individuals, dose 0.1%: 48 individuals.

Details on study design:

The whole study comprised a total subects of 11.357 male and female with both normal and dermatoses.

Irritation parameter:

other: erythema/eschar, oedema, inflammation

Basis:

other: 30 subjects

Remarks:

Test substance concentration 5%

Time point:

24 h

Score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

other: erythema/eschar, oedema, inflammation

Basis:

other: 30 subjects / Test subst concentration 2%

Time point:

other: 24-72 h

Score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

other: erythema/eschar, oedema, inflammation

Basis:

other: 48 subjects

Remarks:

Test substance material 0.1%

Time point:

24/48 h

Score:

0

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

None

Other effects:

No effects

Interpretation of results:

other: not classified

Remarks:

Based on CLP criteria (EC 1272/2008 and its updates)

Conclusions:

Under the conditions of this study, the testing material gave no instances of irritation. Based on this result, the test substance does not need to be classified for skin irritation in accordance with the criteria outlined in CLP (1272/2008/EC).

Executive summary:

The skin irritating potential of Petitgrain Oil on humans was tested in a patch test in 3 concentrations (5, 2 and 0.1%) on human skin at respectively 30, 30, and 48 subjects with and without dermatoses. Under the conditions of this study, the testing material gave no instances of irritation. Based on this result, the test substance does not need to be classified for skin irritation in accordance with the criteria outlined in CLP (1272/2008/EC).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 Sep 2017 - 03 Oct 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

26 July, 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 04455//PGP7216
- Expiration date of the lot/batch: 30 March 2019
- Purity test date: 15 dec 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: Not available

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was tested neat.

OTHER SPECIFICS: UVCB

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Vitelco slaughterhouse, 's Hertogenbosch, The Netherlands
- Characteristics of donor animals (e.g. age, sex, weight): young cattle
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Eyes were collected and transported in physiological saline in a suitable container under cooled conditions
- Time interval prior to initiating testing: as soon as possible after slaughter
- Indication of any existing defects or lesions in ocular tissue samples: The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µl of either the negative control, positive control (Ethanol) or test item.
- Concentration (if solution): Undiluted.

Duration of treatment / exposure:

Corneas were incubated in a horizontal position for 10 ± 1 minutes at 32 ± 1 °C. After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Earle’s Minimum Essential Medium, Life Technologies) and thereafter with cMEM.

Duration of post- treatment incubation (in vitro):

Corneas were incubated for 120 ± 10 minutes at 32 ± 1°C.

Number of animals or in vitro replicates:

triplacates

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded. The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-gl utamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1°C. The corneas were incubated for the minimum of 1 hour at 32 ± 1°C.

QUALITY CHECK OF THE ISOLATED CORNEAS
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (BASFOP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

NUMBER OF REPLICATES
Three corneas were selected at random for each treatment group. 2 experiments were performed.

NEGATIVE CONTROL USED
Physiological saline (Eurovet Animal Health, Bladel, The Netherlands).

POSITIVE CONTROL USED
Ethanol, purity >99.9%

APPLICATION DOSE AND EXPOSURE TIME
750 µl undiluted test item was introduced onto the epithelium of the cornea. Corneas were incubated in a horizontal position for 10 ± 1 minutes at 32 ± 1°C.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Earle’s Minimum Essential Medium, Life Technologies) and thereafter with cMEM. Possible pH effects of the test item on the corneas were recorded.
POST-EXPOSURE INCUBATION: Corneas were incubated for 120 ± 10 minutes at 32 ± 1 °C with fresh cMEM.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity of a cornea was measured by the diminution of light passing through the cornea.The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of a microplate reader (TECAN Infinite® M200 Pro Plate Reader).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS) = mean opacity value + (15 x mean OD490 value).

DECISION CRITERIA:
The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score: In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value) Additionally the opacity and permeability values were evaluated independently to determine whether the test item induced irritation through only one of the two endpoints. The IVIS cut-off values for identifying the test items as inducing serious eye damage (UN GHS Category 1) and test items not requiring classification for eye irritation or serious eye damage (UN GHS No Category): In vitro score range ≤ 3: No Category, > 3 to ≤ 55: No prediction can be made, >55: Category 1.

Irritation parameter:

in vitro irritation score

Run / experiment:

First experiment

Value:

3.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

1.4

Positive controls validity:

valid

Remarks:

5

Irritation parameter:

in vitro irritation score

Run / experiment:

Second experiment

Value:

5.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0.4

Positive controls validity:

valid

Remarks:

57

Irritation parameter:

cornea opacity score

Run / experiment:

First experiment

Value:

4.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

1.2

Positive controls validity:

valid

Remarks:

18

Irritation parameter:

cornea opacity score

Run / experiment:

Second experiment

Value:

5.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0.4

Positive controls validity:

valid

Remarks:

22

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (Ethanol) was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Historical control data

	Negative control			Positive control
	Opacity	Permeability	In vitro Irritancy Score	In vitro Irritancy Score
Range	-2.9 – 3.0	-0.016 – 0.042	-2.8 – 3.0	34.7 – 78.2
Mean	0.08	0.01	0.17	56.01
SD	1.04	0.01	1.14	12.51
n	84	77	78	55

SD = Standard deviation

n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of Aug 2014 to Aug 2017.

Interpretation of results:

other: No prediction on classification can be made

Remarks:

Based on CLP criteria (EC 1272/2008 and its updates)

Conclusions:

Under the conditions of this study, Petitgrain Oil (citrus aurantium) induced an IVIS > 3 ≤ 55, therefore no prediction on the classification can be made in accordance with the criteria outlined in Annex I of the CLP regulations (1272/2008/EC).

Executive summary:

The eye irritation potential of Petitgrain oil (citrus aurantium) was measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test). The study procedures described in this report were based on the most recent OECD test guideline 437. Petitgrain oil (citrus aurantium) was applied as received to the test system, and induced a mean in vitro irritancy score of 3.9 (first experiment) and 5.8 (second experiment) after 10 minutes of treatment. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (Ethanol) was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. Under the conditions of this study, Petitgrain Oil (citrus aurantium) induced an IVIS > 3≤55, therefore no prediction on the classification can be made in accordance with the criteria outlined in Annex I of the CLP regulations (1272/2008/EC).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Main constituents classification

Petitgrain oil - citrus aurantum (all qualities) has two main consituents Linalyl Acetate (CAS 115 -95 -7) and Linalool (CAS 78 -70 -6) with typical concentration values sum up to 50 -90%. Both constituents are classified as category 2 skin and eye irritant in accordance with the criteria outlined in CLP (1272/2008/EC).

Skin irritation

Kligman_1976_KEY_human
The skin irritating potential of Petitgrain Bigarde Oil on humans was tested on 25 healthy adult volunteers. The material was applied under occlusion on the volar aspects of the forearm or backs for 48 hours. No subject had any irritation from the testing material. Under the conditions of this study, the testing material gave no instances of irritation. Based on this result, the test substance does not need to be classified for skin irritation in accordance with the criteria outlined in CLP (1272/2008/EC).

Fujii_1972_Supporting_Human

The skin irritating potential of Petitgrain Oil on humans was tested in a patch test in 3 concentrations (5, 2 and 0.1%) on human skin at respectively 30, 30, and 48 subjects with and without dermatoses. Under the conditions of this study, the testing material gave no instances of irritation. Based on this result, the test substance does not need to be classified for skin irritation in accordance with the criteria outlined in CLP (1272/2008/EC).

Urbach_1976_SUP_mouse

In a dermal irritation study, irritation was evaluated at the non-irradiated sites in an associated phototoxicity study conducted on 15 hairless mice with neat Petitgrain Bigarde Oil. No irritation was observed. The substance did not show any irritant, skin staining or phototoxic effects. Under the conditions of this test, Petitgrain Bigarde Oil is not an irritant to skin in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).

Urbach_1976_SUP_pig

In a dermal irritation study, irritation was evaluated at the non-irradiated sites in an associated phototoxicity study conducted on 5 miniature swine with neat Petitgrain Bigarde Oil. No irritation was observed. The substance did not show any irritant, skin staining or phototoxic effects. Under the conditions of this test, Petitgrain Bigarde Oil is not an irritant to skin in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).

Eye irritation
The eye irritation potential of Petitgrain oil (citrus aurantium) was measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test). The study procedures described in this report were based on the most recent OECD test guideline 437. Petitgrain oil (citrus aurantium) was applied as received to the test system, and induced a mean in vitro irritancy score of 3.9 (first experiment) and 5.8 (second experiment) after 10 minutes of treatment. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (Ethanol) was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. Under the conditions of this study, Petitgrain Oil (citrus aurantium) induced an IVIS > 3≤55, therefore no prediction on the classification can be made in accordance with the criteria outlined in Annex I of the CLP regulations (1272/2008/EC).

Justification for classification or non-classification

Petitgrain oil - citrus aurantum has two main constituents Linalyl Acetate (CAS 115 -95 -7) with concentration range values 40 -68 % (w/w) and Linalool (CAS 78 -70 -6) with concentration range values 10 -35% (w/w). Both constituents are classified as Category 2 skin irritant and eye irritant. Despite no significant effects for skin irritation were found in tests with Petitgrain Oil on humans, pig and mouse under the conditions of those tests, Petitgrain oil - citrus aurantum is classified as Skin Irritant Category 2 based on the classification of these two main constituents. The Eye irritating potential of Petitgrain Oil was assessed with a corneal opacity and permeability test and effects of eye irritation were found. Classification as Eye Irritant Category 2 is based on the classification of the two main constituents as eye irritant and supported by the borderline results of the eye irritation test, making this classification a worst-case conclusion.

Petitgrain oil - citrus aurantum should be classified as skin irritant (Skin Irrit. 2 / H315) and eye irritant (Eye Irrit. 2 / H319) in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).