Essential oil of Copaiba obtained from the exudate of Copaifera (Fabaceae) trees by distillation

Endpoint summary

Description of key information

Skin irritation (OECDTG439): not irritant

Eye irritation (OECDTG437): not irritant

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24-04-2017 to 01-05-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

28 July 2015

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

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: Obtained from sponsor, batch S-71699
- Expiration date of the lot/batch: 14 February 2019
- Purity test date: 14 February 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from light

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2)

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2)
- Tissue batch number(s): 17-EKIN-017
- Production date: 25 April 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37.0 ± 1.0°C (36.3 - 37.4°C)

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: after the exposure period, the tissues were washed with phosphate buffered saline to remove residual test item.
- Observable damage in the tissue due to washing: not reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT concentrate (Sigma Aldrich, Zwijndrecht, The Netherlands; 3 mg/ml in PBS) diluted (10x) in Assay medium (final concentration 0.3 mg/ml).
- Incubation time: 3 h at 37°C
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the
laboratory historical control data range.
- The standard deviation value of the percentage viability of three tissues treated identically was less than 18%, indicating that the test system functioned properly.

NUMBER OF REPLICATE TISSUES: 3

DECISION CRITERIA
- The test substance is considered to be irritant to skin if the relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
- The test substance is considered to be non-irritant to skin if the relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 25μL undiluted
NEGATIVE CONTROL
- Amount(s) applied: 25μL undiluted
POSITIVE CONTROL
- Amount(s) applied: 25μL
- Concentration (if solution): 5%

Duration of treatment / exposure:

15 ± 0.5 minutes

Duration of post-treatment incubation (if applicable):

42 hours at 37°C

Number of replicates:

three

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Main experiment in triplicate

Value:

107

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

20%

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: not reported
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 20%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 18%, indicating that the test system functioned properly.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline:
Negative control: Absorption OD570=0.676-1.336, Mean=1.01, SD=0.016, n=155
Positive control: Absorption OD570=0.036-0.549, Mean=0.16, SD=0.10, n=154
Positive control: Viability %= 2.85-45.43, Mean=15.74, SD=9.22, n=163

Interpretation of results:

other: not classified

Remarks:

based on CLP criteria (Annex I 1272/2008/EC)

Conclusions:

Based on the results obtained, it can be concluded that Copaiba balsam oil is not considered a skin irritant, and does not have to be classified for skin irritation in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).

Executive summary:

The skin irritation potential of Copaiba balsam oil was tested in accordance to OECD TG 439. Undiluted Copaiba balsam oil was topically applied to EPISKIN-SMTM for 15 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed using MTT conversion measurements. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with Copaiba balsam oil compared to the negative control tissues was 107%. Since the mean relative tissue viability for Copaiba balsam oil was above 50% after treatment, the testing material it is not considered to be an irritant. Both the positive (20% viability) and the negative control were within the historical control data range and therefore considered valid. Furthermore, the standard deviation value of the percentage viability of three tissues treated identically was less than 18%, indicating that the test system functioned properly.

Based on the results obtained, it can be concluded that Copaiba balsam oil is not considered a skin irritant, and does not have to be classified for skin irritation in accordance with the criteria outlined in Annex I of the CLP Regulation (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:

11-04-2017

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

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: Obtained from sponsor, batch S-71699
- Expiration date of the lot/batch: 14 February 2019
- Purity test date: 14 February 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from light

Species:

cattle

Details on test animals or tissues and environmental conditions:

Test System
- Source: Vitelco slaughterhouse, 's Hertogenbosch, The Netherlands
- Age at study initiation: young cattle
- Other info: the eyes were excised by a slaughterhouse employee as soon as possible after slaughter. Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.

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 or undiluted test item was introduced onto the epithelium of the cornea.

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 with cMEM.

Number of animals or in vitro replicates:

Triplicate

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 containing 1% (v/v) L-glutamine and 1% (v/v) Foetal Bovine Serum ). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF 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:
-Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0,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: 3
NEGATIVE CONTROL USED:
- A negative control, physiological saline (Eurovet Animal Health, Bladel, The Netherlands) was included to detect non-specific changes in the test system and to provide a baseline for the assay endpoints.
POSITIVE CONTROL USED:
- Ethanol, Batch K47177483, Identification number RS532, Purity >99.9% Stable under storage conditions until 31 October 2020
APPLICATION DOSE AND EXPOSURE TIME:
- Undiluted, 10 +/- 1 minutes at 32 +/- 1°C
TREATMENT METHOD:
- The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF 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.
REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 2
- POST-EXPOSURE INCUBATION: yes; 120 +/- 10 minutes at 32 +/- 1°C.
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)
ACCEPTABILITY CRITERIA
The assay is considered acceptable if:
1) The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean.
2) The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

EVALUATION 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) are: ≤ 3: No Category, > 3 ≤ 55: No prediction can be made, >55: Category 1

Irritation parameter:

in vitro irritation score

Run / experiment:

Main experiment

Value:

ca. -0.7 - -0.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: not reported
DEMONSTRATION OF TECHNICAL PROFICIENCY:
- 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 61 and 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.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values if different from the ones specified in the test guideline:

Negative control
-opacity -2.9-3.0 (SD 1.07, n=72)
-permeability -0.016-0.042 (SD 0.01, n=65)
-IVIS -2.8-3.0 (SD 1.17, n=66)
Positive control:
-IVIS 34.7-78.2 (SD 12.64, n=47)

Summary of Opacity, Permeability and In Vitro Scores

Treatment	Mean Opacity (1)	Mean Permeability (1)	Mean In vitro Irritation Score (1,2)
Negative control	-0.5	0.003	-0.4
Positive control (Ethanol)	19.6	2.737	60.7
Copaiba balsam oil	-0.5	0.009	-0.4

(1) Calculated using the negative control mean opacity and mean permeability values for the positive control and test item.

(2) In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).

Interpretation of results:

other: not classified

Remarks:

based on CLP criteria (Annex I of 1272/2008/EC)

Conclusions:

Copaiba balsam oil induced an IVIS ≤ 3. Based on these results, the test substance does not need to be classified as eye irritant according to the classification criteria outlined in Annex I of 1272/2008/EC (CLP).

Executive summary:

The eye hazard potential of Copaiba balsam oil was evaluated according to OECD guideline 437 (BCOP test). The eye damage was assessed through topical application of 750 μl of the undiluted Copaiba balsam oilfor 10 minutes on top of the corneas. Both the negative control and the positive control (Ethanol) were considered valid. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. Copaiba balsam oil did not induce ocular irritation (no opacity and no permeability), resulting in a mean in vitro irritancy score of -0.4 after 10 minutes of treatment.

In conclusion, since Copaiba balsam oil induced an IVIS ≤ 3, and the test substance does not need to be classified as eye irritant according to the classification criteria outlined in Annex I of 1272/2008/EC (CLP).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Skin irritation in vitro

The skin irritation potential of Copaiba balsam oilwas tested in accordance to OECD TG439. Undiluted Copaiba balsam oil was topically applied to EPISKIN-SMTM for 15 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed using MTT conversion measurements.The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with Copaiba balsam oil compared to the negative control tissues was 107%. Since the mean relative tissue viability for Copaiba balsam oil was above 50% after treatment, the testing material it is not considered to be an irritant. Both the positive (20% viability) and the negative control were within the historical control data range and therefore considered valid. Furthermore, the standard deviation value of the percentage viability of three tissues treated identically was less than 18%, indicating that the test system functioned properly. Based on the results obtained, it can be concluded that Copaiba balsam oil is not considered a skin irritant, and does not have to be classified for skin irritation in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).

Eye irritation in vitro

The eye hazard potential of Copaiba balsam oil was evaluated according to OECD guideline 437 (BCOP test). The eye damage was assessed through topical application of 750 μl of the undiluted Copaiba balsam oil for 10 minutes on top of the corneas. Both the negative control and the positive control (Ethanol) were considered valid. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. Copaiba balsam oil did not induce ocular irritation (no opacity and no permeability), resulting in a mean in vitro irritancy score of 0.7 after 10 minutes of treatment. In conclusion, since Copaiba balsam oil induced an IVIS ≤ 3, and the test substance does not need to be classified as eye irritant according to the classification criteria outlined in Annex I of 1272/2008/EC (CLP).

Justification for classification or non-classification

Based on the available information, the test substance does not need to be classified for skin irritation and eye irritation, according to the classification criteria outlined in Annex I of 1272/2008/EC (CLP).