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Administrative data

Description of key information

Skin irritation using read across from Schinus Terebinthifolius (OECD TG 439): irritating (based on evaluation of constituent information and study data)

Eye irritation (OECD TG 438): not irritating

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11-29 May 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
adopted 28 July 2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
23 July 2009
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
UK GLP Compliance Program (inspected on July 05, 2016 / Signed on October 28, 2016)
Specific details on test material used for the study:
Storage conditions: Room temperature in the dark until 19 January 2017 thereafter approximately 4°C in the dark
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
Following the REACH top-down strategy, the EPISKIN™ Reconstructed Human Epidermis Model method was used to assess skin irritation as recommended in the OECD test guideline No. 439.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ Reconstructed Human Epidermis Model Kit, SkinEthic Laboratories, Lyon, France
- Tissue batch number(s): 17-EKIN-019
- Production date: not reported
- Shipping date: not reported
- Delivery date: 10 May 2017
- Expiry date: 15 May 2017
- Date of initiation of testing: 11 May 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each wellsidual test or control items and then incubated in 2mL maintenance medium for 42 h at 37 °C, 5 % CO2 in air. Number of washing steps not reported.
- Observable damage in the tissue due to washing: none reported
- Modifications to validated SOP:
Deviation 1
Due to unusual variation noted in the untreated killed control group, it was considered necessary to repeat the killed tissue groups.
Deviation 2
An assessment found the test item was able to directly reduce MTT. Therefore, an additional procedure using water killed tissues was performed. However, the results obtained showed that no interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the water killed tissues for quantitative correction of results or for reporting purposes.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Labtech LT 4500 microplate reader
- Wavelength: 570 nm
- Filter: without reference filter
- Linear OD range of spectrophotometer: not reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Barrier function: IC50 = 1.8 mg/ml ( ≥ 1.5 mg/ml)
- Morphology: Well-differenciated epidermis consisting of a basal layer, several spinous and granular layers and a thinck stratum corneum
- Contamination: absence of bacteria, fungus and mycoplasma
- Reproducibility: All of the values for the negative and positive control groups fell within the historical ranges of the testing laboratory obtained in the previous six months. This was taken to show the correct functioning of the test system.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE:
- Fresh tissues / killed tissues: skin irritation potential performed in parallel on viable and water killed tissues for quantitative correction of the results
- Procedure used to prepare the killed tissues (if applicable): Water-killed tissues were prepared prior to the study by placing untreated EPISKINTM tissues in a 12 well plate containing 2.0 mL of sterile distilled water in each well. The tissues were incubated at 37°C, 5% CO2 in air for 48 +/- 1 hours. At the end of the incubation the water was discarded. Once killed the tissues were stored in a freezer (-14 to +30 °C) for up to 6 months. Before use each tissue was thawed by placing in 2.0 mL of maintenance medium for approximately 1 hour at room temperature.
- N. of replicates : 3 (test item + untreated)
- Method of calculation used: the results obtained showed that no interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the water killed tissues for quantitative correction of results or for reporting purposes.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritating to skin if relative mean tissue viability is ≤ 50% after 15 minutes of exposure.
- The test substance is considered to be non-irritating to skin if relative mean tissue viability is > 50% after 15 minutes of exposure.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- The test item was used as supplied (undiluted).
- Amount(s) applied (volume or weight with unit): 10 ± 2 mg of the test substance (powder) was dispensed over each tissue. The tissues were wetted with 5 μL of purified water prior to application of the test substance.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL
- Concentration (if solution): DPBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL
- Concentration (if solution): Sodium Dodecyl Sulphate (SDS) at a 5% (w/v) aqueous solution
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
Triplicate tissues for test substance, negative and positive controls
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
15 minute exposure period and 42 h post-exposure incubation period
Value:
95.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
12.2%
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: none reported
- Direct-MTT reduction: yes, an assessment found the test item was able to directly reduce MTT. Therefore, an additional procedure using water killed tissues was performed. However, the results obtained showed that no interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the water killed tissues for quantitative correction of results or for reporting purposes.
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD570 for the negative control treated tissues was 0.656 and the standard deviation value of the viability was 7.3%.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 12.2% relative to the negative control treated tissues and the standard deviation value of the viability was 0.7%.
- Acceptance criteria met for variability between replicate measurements: The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 11.5%.
- Reference to historical values: All of the values for the negative and positive control groups fell within the historical ranges of the testing laboratory obtained in the previous six months. This was taken to show the correct functioning of the test system.

Table 7.3.1/1: EpiSkin™ results

Item

OD570of tissues

Mean OD570of triplicate tissues

±SDof OD570

Relative individual tissue viability (%)

Relative mean viability (%)

± SD of Relative mean viability (%)

Negative Control Item

0.661

0.656

0.048

100.8

100*

7.3

0.701

106.9

0.606

92.4

Positive Control Item

0.084

0.080

0.005

12.8

12.2

0.7

0.081

12.3

0.075

11.4

Test Item

0.549

0.629

0.076

83.7

95.9

11.5

0.640

97.6

0.699

106.6


OD=Optical Densit

SD=       Standard deviatio

*=        The mean viability of the negative control tissues is set at 100%

Interpretation of results:
other: not skin irritating
Remarks:
in accordance with EU CLP (EC 1272/2008 and its updates)
Conclusions:
Under the experimental conditions of this study, the test substance is not classified for skin irritation according to Regulation (EC) No. 1272/2008 (CLP) and to the GHS.
Executive summary:

An in vitro skin irritation study was performed according to the OECD Guideline 439 and in compliance with GLP, using the EPISKINTM reconstructed human epidermis model.

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. The test item was found to directly reduce MTT and therefore additional non-viable tissues were incorporated into the testing for correction purposes. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density was measured at 570 nm.

 

The relative mean viability of the test item treated tissues was 95.9% after the 15-Minute exposure period and 42-Hours post-exposure incubation period.

The relative mean tissue viability for the positive control treated tissues was 12.2% relative to the negative control treated tissues and the standard deviation value of the viability was 0.7%. The positive control acceptance criteria were therefore satisfied.

The mean OD570 for the negative control treated tissues was 0.656 and the standard deviation value of the viability was 7.3%. The negative control acceptance criteria were therefore satisfied.

The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 11.5%. The test item acceptance criterion was therefore satisfied.

All of the values for the negative and positive control groups fell within the historical ranges of the testing laboratory obtained in the previous six months. This was taken to show the correct functioning of the test system.

 

Under the experimental conditions of this study, the test substance is not skin irritating.

This study is considered as acceptable and satisfies the requirement for skin irritation endpoint.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: read across
Justification for type of information:
The read-across justification is presented in the endpoint summary and the accompanying files are also attached there.
Reason / purpose for cross-reference:
read-across source
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
15 minute exposure period and 42 h post-exposure incubation period
Value:
95.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
12.2%
Remarks on result:
no indication of irritation
Interpretation of results:
other: not irritating
Remarks:
in accordance with EU CLP (EC 1272/2008 and its updates)
Conclusions:
Under the experimental conditions of this study, the Pink pepper oil (source) is not classified for skin irritation according to Regulation (EC) No. 1272/2008 (CLP) and to the GHS. This result was used for read-across to Schinus molle oil (target).
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (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:
18 Jan 2018 to 02 Feb 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 438 (Isolated Chicken Eye 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:
adopted on 9 October 2017
GLP compliance:
yes (incl. QA statement)
Remarks:
Triskelion B.V., Utrechtseweg 48, 3704 HE Zeist, The Netherlands
Species:
other: Eyes of male or female chickens (ROSS, spring chickens)
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Slaughterhouse v.d. Bor, Nijkerkerveen, The Netherlands
- Characteristics of donor animals: Approximately 7 weeks old, male or female chickens, body weight range approximately 1.5-2.5 kg, were used as eye donors.
- Storage, temperature and transport conditions of ocular tissue: Heads of the animals were cut off immediately after sedation of the animals by electric shock and incision of the neck for bleeding, and before they reached the next station on the process line. The heads were placed in small plastic boxes on a bedding of paper tissues moistened with isotonic saline. Next, they were transported to the testing facility. During transportation, the heads were kept at ambient temperature.
- Time interval prior to initiating testing: Within 2 hours after kill, eyes were carefully dissected and placed in a superfusion apparatus.
- Indication of any existing defects or lesions in ocular tissue samples: No
- Indication of any antibiotics used: No
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
30 μL neat substance
Duration of treatment / exposure:
10 seconds
Duration of post- treatment incubation (in vitro):
240 minutes
Number of animals or in vitro replicates:
Test group and positive control: triplicates
Negative control: Single
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES
Within 2 hours after kill, eyes were carefully dissected and placed in a superfusion apparatus using the following procedure: First the eye-lids were carefully removed without damaging the cornea and a small drop of Fluorescein sodium 2.0 % w/v was applied to the corneal surface for a few seconds and subsequently rinsed off with isotonic saline at ambient temperature. Next, the head with the fluorescein-treated cornea was examined with a slit-lamp microscope (Slit-lamp 900 BP, Haag-Streit AG, Liebefeld-Bern, Switzerland) to ensure that the cornea was not damaged. If undamaged (e.g., fluorescein retention and corneal opacity scores of ≤ 0.5), the eye was further dissected from the head without damaging the eye or cornea. Care was taken to remove the eye-ball from the orbit without cutting off the optical nerve too short. The enucleated eye was placed in a stainless steel clamp with the cornea positioned vertically and transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with isotonic saline from a bent, stainless steel tube, at a target rate of 0.10-0.15 mL/min. The chambers of the superfusion apparatus as well as the saline were temperature controlled at approximately 32 °C (water pump set at 36.4 °C). After placing in the superfusion apparatus, the eyes were examined again with the slit-lamp microscope to ensure that they were not damaged. An accurate measurement was taken at the corneal apex of each eye. Eyes with a corneal thickness deviating more than 10 % of the average corneal thickness of the eyes, eyes showing opacity (score higher than 0.5), or were unacceptably stained with fluorescein (score higher than 0.5) indicating the cornea to be permeable, or eyes that showed any other signs of damage, were rejected as test eyes and replaced.

EQUILIBRATION AND BASELINE RECORDINGS
Each eye provided its own baseline values for corneal swelling, corneal opacity and fluorescein retention. For that purpose, after an equilibration period of 45-60 minutes, the corneal thickness of the eyes was measured again to determine the zero reference value for corneal swelling calculations.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: 20 mL saline. After rinsing, each eye in the holder was returned to its chamber.
- Indicate any deviation from test procedure in the Guideline: none

METHODS FOR MEASURED ENDPOINTS
- Corneal opacity: Slit-lamp microscope examination
- Damage to epithelium based on fluorescein retention: Slit-lamp microscope examination
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting: set at 0.095 mm
- Others: After the final examination, the test substance treated eyes, the negative and positive control eyes were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde. The corneas were embedded in paraffin wax, sectioned at ca 4 μm and stained with PAS (Periodic Acid-Schiff). The microscopic slides were subjected to histopathological examination.

SCORING SYSTEM
Defined scoring scales were used for each parameter to define the severity of effects into four categories (I-IV).
- Mean corneal swelling (%): According to OECD 438 guideline. Examination of the eyes after 0, 30, 75, 120, 180, and 240 minutes
- Mean maximum opacity score: According to OECD 438 guideline. Examination of the eyes after 0, 30, 75, 120, 180, and 240 minutes
- Mean fluorescein retention score at 30 minutes post-treatment: According to OECD 438 guideline.

DECISION CRITERIA
According to OECD 438 guideline
Irritation parameter:
percent corneal swelling
Run / experiment:
slit-lamp examination
Value:
2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: maximum mean values
Irritation parameter:
cornea opacity score
Run / experiment:
slit-lamp examination
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: maximum mean values
Irritation parameter:
fluorescein retention score
Run / experiment:
slit-lamp examination
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: maximum mean values
Other effects / acceptance of results:
- Slit-lamp examination: The substance caused very slight corneal swelling (mean score 2 %). The negative control eye did not show any corneal effect and demonstrated that the general conditions during the test were adequate. The positive control BAC 5 % caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants.
- Microscopic examination: Microscopic examination of the corneas treated with the test substance did not reveal any abnormalities. Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities. Microscopic examination of the corneas treated with the positive control BAC 5 % generally revealed severe erosion and very slight or slight vacuolation of the epithelium and endothelial necrosis.
Interpretation of results:
other: Not irritating
Remarks:
Based on CLP criteria (EC 1272/2008 and its updates)
Conclusions:
Under the test conditions (OECD TG 438 and GLP) the test substance is not considered to be an eye irritant in accordance with the criteria outlined in Annex I of the CLP regulations (1272/2008/EC).
Executive summary:

The test substance was examined for its in vitro eye irritating potential using the Isolated Chicken Eye (ICE) test, in accordance with OECD guideline 438 and GLP. In the ICE test, 3 eyes were exposed to 30 µL neat test substance for 10 seconds. In addition, one negative control eye (30 µL saline) and three positive control eyes (30 µL 5 % Benzalkonium Chloride (BAC)) were tested. After the exposure, the eyes were rinsed with 20 mL saline and were examined at approximately 0, 30, 75, 120, 180, and 240 minutes after treatment. Mean fluorescein retention score was determined at 30 minutes post-treatment. The substance caused very slight corneal swelling (mean score 2 %). The negative control eye did not show any corneal effect and demonstrated that the general conditions during the test were adequate. The positive control BAC 5 % caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants. Microscopic examination of the corneas treated with the test substance did not reveal any abnormalities. Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities. Microscopic examination of the corneas treated with the positive control BAC 5 % generally revealed severe erosion and slight vacuolation of the epithelium and endothelial necrosis. Based on these results, classification of the substance for eye irritation is not warranted.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Additional information

Eye irritation in vitro

The test substance was examined for its in vitro eye irritating potential using the Isolated Chicken Eye (ICE) test, in accordance with OECD guideline 438 and GLP. In the ICE test, 3 eyes were exposed to 30 µL neat test substance for 10 seconds. In addition, one negative control eye (30 µL saline) and three positive control eyes (30 µL 5 % Benzalkonium Chloride (BAC)) were tested. After the exposure, the eyes were rinsed with 20 mL saline and were examined at approximately 0, 30, 75, 120, 180, and 240 minutes after treatment. Mean fluorescein retention score was determined at 30 minutes post-treatment. The substance caused very slight corneal swelling (mean score 2 %). The negative control eye did not show any corneal effect and demonstrated that the general conditions during the test were adequate. The positive control BAC 5 % caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants. Microscopic examination of the corneas treated with the test substance did not reveal any abnormalities. Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities. Microscopic examination of the corneas treated with the positive control BAC 5 % generally revealed severe erosion and slight vacuolation of the epithelium and endothelial necrosis. Based on these results, classification of the substance for eye irritation is not warranted.

 

Skin irritation (read across from Schinus Terebinthifolius)

The skin irritation of Schinus Molle oil was assessed by using constituent information and read across from Schinus Terebinthifolius (CAS No.: 949495-68-5). First the experimental information of the source substance will be summarised. Thereafter the read across justification is presented. The accompanying files are attached in the present endpoint summary. Taking together the negative result of the source study and the uncertainty of the read-across as described in the justification, the target substance is considered to be a skin irritant.

 

Skin irritation in vitro

An in vitro skin irritation study was performed according to the OECD Guideline 439 and in compliance with GLP, using the EPISKIN reconstructed human epidermis model. Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. The test item was found to directly reduce MTT and therefore additional non-viable tissues were incorporated into the testing for correction purposes. At the end of the postexposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to prelabelled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTTloaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µL samples were transferred to the appropriate wells of a prelabelled 96well plate. The optical density was measured at 570 nm. The relative mean viability of the test item treated tissues was 95.9% after the 15Minute exposure period and 42Hours postexposure incubation period. The relative mean tissue viability for the positive control treated tissues was 12.2% relative to the negative control treated tissues and the standard deviation value of the viability was 0.7%. The positive control acceptance criteria were therefore satisfied. The mean OD570 for the negative control treated tissues was 0.656 and the standard deviation value of the viability was 7.3%. The negative control acceptance criteria were therefore satisfied. The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 11.5%. The test item acceptance criterion was therefore satisfied. All of the values for the negative and positive control groups fell within the historical ranges of the testing laboratory obtained in the previous six months. This was taken to show the correct functioning of the test system. Under the experimental conditions of this study, Pink Pepper is considered not skin irritating.

 

Read-across justification

Schinus Molle oil (CAS 94334-31-3; target) and its irritating properties using read across from Essential oil of Schinus Terebinthifolius (Anacardiaceae) obtained from red berries by supercritical carbon dioxide extraction (indicative), hereafter named Pink Pepper oil (CAS 949495-68-5; source)

 

Introduction and hypothesis for the read across

Schinus Molle oil is a UVCB, containing hydrocarbon constituents. The main constituents have three C=C double bonds (myrcene), a 6 ring with two C=C double bonds (alpha-phellandrene), a 6 ring with a C=C double bond inside and a C=C double bond outside the ring (DL-Limonene), a 6 ring with a C=C double bond inside and a C=C double bond outside the ring (beta-phellandrene). For Schinus Molle oil (target) no skin irritation data are available. Therefore additional information is used in accordance with Article 13 of REACH where it is said that lacking information should be generated whenever possible by means other than vertebrate animal tests, i.e. applying alternative methods such as in vitro tests, SARs, grouping and read-across. For assessing the skin irritation of Schinus Molle oil, the irritation data of Pink Pepper oil is used.

 

Hypothesis:Schinus Molle oil (target) is expected to have equivalent irritating properties based on information from Pink Pepper oil (source) because both have common constituents in comparable concentrations in the oil.

 

Available skin irritation information:

The source substance Pink Pepper oil (Essential oil of Schinus Terebinthifolius (Anacardiaceae) obtained from red berries by supercritical carbon dioxide extraction) has been tested in a well performedOECD TG 439 study, under GLP. The skin irritation potential of the test item was assessed using the EPISKINTMreconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. After exposure, the viability and standard deviations between the triplicates of both the positive control and the negative control were within the acceptance criterion. The relative mean viability of the test item treated tissues was 95.9%. The test item was therefore considered a non-irritant.

 

Target and Source chemical(s):

The composition of the target chemical (Schinus Molle oil) and the source chemical (Pink Pepper oil) are shown in Data matrix 1. Schinus Molle oil and Pink Pepper oil are both substances of Unknown or Variable Composition, Complex Reaction Products and Biological Materials (UVCBs), obtained by steam distillation from the Berries from Schinus Molle tree (Anacardiaceae) and by supercritical carbon dioxide extraction (indicative) from the Schinus Terebinthifolius red berries, respectively.

 

Similarities in composition:

The target and the source share nine common constituents: Pin-2(3)-ene, Thuj-4(10)-ene, Pin-2(10)-ene, 7-methyl-3-methyleneocta-1,6-diene, p-mentha-1,5-diene, para-cymene, Dipentene, p-mentha-1(7),2-diene and beta-caryophyllene.

Schinus Molle oil and Pink Pepper oil have a comparable amount of minors/unknowns: 10.00-25.00% w/w present in Schinus Molle oil and 0.01-20.00% w/w present in Pink pepper.

 

Differences in composition:

- p-mentha-1,5-diene (Alpha-phellandrene) and 7-methyl-3-methyleneocta-1,6-diene (myrcene) are present in higher amounts in Schinus Molle oil (target) than in Pink Pepper oil (Source).

- The following two constituents (> 1%) are only present in Schinus Molle oil (target): (+)-Delta cadinene (1.0-5.00%) and Alpha muurolene (0.5-3.00%).

- The following seven constituents (> 1%) are only present in Pink Pepper oil (source): Delta 3-carene (1.00-29.00%), Germacrene D (3.00-14.00%), Germacrene B (0.01-3.00%), Elemol (0.01-6.00%), Spathulenol (0.00-3.00%), Bicyclogermacrene (0.01-3.00%) and Terpinolene (0.01-3.00%).

 

The focus will be on the common constituents that are present in both Schinus Molle oil and Pink Pepper oil, as well as the constituents present in Schinus Molle oil but not in Pink Pepper oil. The information on Schinus Molle oil (target) and the information from Pink Pepper oil (source) are presented in the data matrices below. This includes physico-chemical properties and toxicological information, relevant for irritation.

 

Purity / Impurities:

The impurities are not relevant for Schinus Molle oil (target) and Pink Pepper oil (source) as they are both UVCBs, however a constituent profile is applicable. For target and source the constituents are presented in the data matrices below. The similarity and differences in constituents between target and source will be discussed below in the analogue justification.

 

Analogue justification

According to Annex XI 1.5, read across can be used to replace testing when the similarity between the substances based on composition can be established. When using read across the result derived should be applicable for C&L and/or risk assessment and it should be presented with adequate and reliable documentation.

 

Analogue selection:

Pink Pepper oil was selected as analogue because of the similarity in constituents with Schinus Molle oil (see Data matrix 2). Pink Pepper oil and Schinus Molle oil have many constituents in common in comparable amounts which justifies the analogue approach. The constituents that are present in significant concentrations in Schinus Molle oil but not (or in a lower concentration range) in Pink Pepper oil will be addressed in the toxico-dynamic section.

 

Bioavailability:Dermal absorption of both UVCBs is expected despite variation in properties of some constituents. The molecular weight and liquid appearance of the constituents in both UVCBs are favorable for dermal absorption. Based on the water solubility and log Kow of all constituents, absorption is anticipated to be low to moderate. However due to the generally high LogKow (>4) of the majority of constituents the rate of penetration may be limited by the rate of transfer between the stratum corneum and the epidermis, but uptake into the stratum corneum will be high.

 

Toxico-dynamics:Reactivity is the key parameter for assessing the skin irritating potential. Both Schinus Molle and Pink Pepper are expected to have the comparable reactivity based on the similarity in irritating constituents (a.o. Pin-2(3)-ene (DL-alpha-pinene), Pin-2(10)-ene (Beta-pinene), 7-methyl-3-methyleneocta-1,6-diene (Myrcene), Para-cymene, Dipentene (Limonene), Delta 3-carene) present in both UVCBs.

Two of the known irritating constituents (according to the ECHA Disseminated dossier) are present in a lower amount in the target than in the source; Pin-2(3)-ene (alpha pinene,2-8% vs. 8-23%), delta-3-carene (0% vs 1-29%) which could indicate a greater potential for irritation of the source UVCB, and thus pose a worst-case situation. On the other hand, the concentration ranges of 7-methyl-3-methyleneocta-1,6-diene (myrcene) is higher in the target substance compared to the source (10-25% vs. 0-5%), myrcene is a known skin irritant according to the ECHA disseminated dossier.

 

Data matrix

The relevant information on constituents, physico-chemical properties and toxicological characteristics are presented in the data matrix in Table 1 and 2.

 

Uncertainty of the predictions:

Schinus Molle oil (target) and Pink Pepper (source) contain many constituents in common, in comparable concentrations. The constituents of Schinus Molle oil that are not present in Pink Pepper (alpha muurolene and delta cadinene) have similar functional groups as the common constituents, and are not expected to have an effect on the skin irritating potential. However, the presence of Myrcene in the target in a possibly 5x higher concentration does result in an uncertainty with regard to the potential skin irritation. While the in vitro skin irritation test results for Pink Pepper oil did not result in a classification for skin irritation, Myrcene is a reported skin irritant in the ECHA disseminated dossier based on a reliable in vitro test. To overcome this uncertainty, classification of the target substance for skin irritation is considered necessary.

 

Conclusions for skin irritation

For Schinus Molle oil skin irritation information is not available, and read across from Pink Pepper oil is used. When using read across the result derived should be applicable for C&L and/or risk assessment and be presented with adequate and reliable documentation. The current document presents such documentation. The source substance has been tested in a well performedOECD TG 439 study, under GLP,based on which it was considered anon-irritant.However due to the uncertainties regarding the presence of the known skin irritant Myrcene in higher concentrations in the target substance, the potential for skin irritation cannot be dismissed.

 

Final conclusion on hazard and application in the risk characterization: Based on the available data and the read across justification, the target substance Schinus Molle oil is considered a skin irritant.

 

Data matrix 1. Information on source and target relevant for assessment of skin irritation potential

 

 

Target

 Source

Name

Schinus Molle oil

Pink Pepper oil*

 

Molecular structure

N/A (UVCB)

N/A (UVCB)

 

CAS

94334-31-3

949495-68-5

 

REACH registration

To be registered (Annex VII)

REACH registered

 

EC Number

305-104-2

481-880-7

Molecular formula

N/A (UVCB)

 

N/A (UVCB)

 

Molecular weight

N/A

N/A

 

Physico-chemical properties

 

 

Appearance

Liquid

 

Liquid

Vapour pressure (Pa)

168.9 (Exp)

Range: 2.6 – 372 for 12/13 constituents, one major constituent (alpha-pinene) has a higher value at 851 Pa

Range for all constituents: 2.6-851(QSAR and experimental data)

Water solubility (mg/L)

36.9 (Exp)

Range: 0.0007 - 7.14 (10/13 constituents have a water solubility < 1 mg/L)

(QSAR and experimental data)

 

Log Kow

4.7 – 5.7 (Exp)

Range: 3.68 - 6.4, only one minor constituent has <4. (QSAR and experimental data)

Human health

 

 

Acute oral tox

LD50 > 5000 mg/kg bw

LD50 > 5000 mg/kg bw

Skin sensitisation

Read-across

Skin Sens. 1B

Skin irritation

Read-across

Not classified

Eye irritation

Not classified

Not classified

Genetic toxicity (Ames)

Not mutagenic

Not mutagenic

Exp =Experimental. Reference: IFF, 2017. The Determination of Physico-Chemical Properties of Schinus Molle oil. Report No. 191244.

QSAR = Quantitative Structure-Activity Relationship

*Physico-chemical, and human health information from Pink Pepper oil are derived from ECHA disseminated dossier (May 2018)

N/A= Not applicable


Data matrix 2. Composition of Target and Source and skin irritation information available for these constituents

NAME

CAS

Target

Target

Source

Source

Endpoint specific classification*

 

 

Min.

Max.

Min.

Max.

 

Pin-2(3)-ene

(DL-alpha-pinene)

80-56-8

2.00%

8.00%

8.00%

23.00%

Skin Irrit. 2

 

Thuj-4(10)-ene

(Sabinene)

3387-41-5

2.00%

8.00%

0.01%

5.00%

Not available

Pin-2(10)-ene

(Beta-pinene)

127-91-3

0.50%

3.00%

0.01%

3.00%

Skin Irrit. 2

 

7-methyl-3-methyleneocta-1,6-diene

(Myrcene)

123-35-3

10.00%

25.00%

0.01%

5.00%

Skin Irrit. 2

 

p-mentha-1,5-diene

(Alpha-phellandrene)

99-83-2

20.00%

35.00%

12.00%

33.00%

Not available

Para-cymene

99-87-6

1.00%

5.00%

0.01%

5.00%

Skin Irrit. 2 (no studies in REACH dossier)

Dipentene (Limonene)

138-86-3

5.00%

15.00%

0.01%

15.00%

Skin Irrit. 2

p-mentha-1(7),2-diene (Beta-phellandrene)

555-10-2

5.00%

15.00%

0.01%

10.00%

Not available

Caryophyllene

(beta-caryophyllene)

13877-93-5/87-44-5

0.50%

4.00%

0.01

6.00%

Not classified

(+)-Delta cadinene

483-76-1

1.00%

5.00%

-

-

Not available

Alpha muurolene

31983-22-9

0.50%

3.00%

-

-

Not available

Delta 3-carene

 

13466-78-9

 

-

-

1.00%

29.00%

Skin Irrit. 2

Germacrene D

37839-63-7

-

-

3.00%

14.00%

Not available

Germacrene B

15423-57-1

-

-

0.01%

3.00%

Not available

Elemol

639-99-6/8024-27-9

-

-

0.01%

6.00%

Not available

Spathulenol

6750-60-3

-

-

0.00%

3.00%

Not available

Bicyclogermacrene

24703-35-3

-

-

0.01%

3.00%

Not available

Terpinolene

586-62-9

-

-

0.01%

3.00%

Not classified

Other minor and unknown constituents

-

10.00%

25.00%

0.01%

20.00%

 

*Human-health information from individual constituents is derived from the available ECHA disseminated dossiers (May 2018)

 

Justification for classification or non-classification

Based on the available information, the substance should be classified for skin irritation (Skin Irrit. 2 / H315) but not for eye irritation, in accordance with the criteria outlined in EU CLP (EC no. 1272/2008 and its amendments).