Registration Dossier

Diss Factsheets

Administrative data

Description of key information

Skin irritation (OECDTG439)

Cedarwood Texas oil distilled - Terpenes (Thujopsene): not irritant

Cedarwood Texas oil distilled - Terpenes (Cedrene): irritant (read-across from Cedarwood Virginia oil)

Skin corrosion (OECDTG439)

Cedarwood Texas oil distilled - Terpenes (Cedrene): not corrosive (read-across from Cedarwood Virginia oil)

Eye irritation (OECDTG437)

Cedarwood Texas oil distilled - Terpenes (Thujopsene): not irritant

Cedarwood Texas oil distilled - Terpenes (Cedrene): not irritant (read-across from Cedarwood Virginia oil)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 Jul 2017 - 21 Jul 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
29 July 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test".
Version / remarks:
Official Journal of the European Union No. L142, 31 May 2008.
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: 1002960562
- Expiration date of the lot/batch: 31 August 2017

STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none, the liquid test item was applied undiluted (50 µl) directly on top of the tissue.
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: MatTek Corporation, Ashland MA, U.S.A.
Source strain:
not specified
Justification for test system used:
Recommended test system in international guidelines (OECD and EC).
Vehicle:
unchanged (no vehicle)
Details on test system:
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature for the 3 hour exposure, 37.0 ± 1.0°C (actual range 36.8 - 37.5°C) for the 1 hour exposure

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: not specified
- Observable damage in the tissue due to washing: none

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Wavelength: 570 nm

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION:
3 minute exposure: duplicates each measured 3x
1 hour exposure: duplicates each measured 3x

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
A test item is considered corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%.
b) In addition, a test item considered non-corrosive (viability >=50%) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test item is decreased below 15%.
A test item is considered non corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%.
b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%.

Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50µL
- Concentration (if solution): undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50µL
- Concentration (if solution): Milli-Q undiluted

POSITIVE CONTROL
- Amount(s) applied (volume or weight): control
- Concentration (if solution): 8N KOH
Duration of treatment / exposure:
3 minutes or 1 hour
Duration of post-treatment incubation (if applicable):
After exposure, the DMEM was replaced by 300 µl MTT-medium and tissues were incubated for 3 hours at 37°C in air containing 5% CO2.
Number of replicates:
2 per timepoint
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minute exposure
Value:
94
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 hour exposure
Value:
103
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: yes, the non-specific reduction of MTT by the test item was -0.46% and 0.29% of the negative control tissues after 3 minutes and 1 hour respectively.
- Colour interference with MTT: yes

DEMONSTRATION OF TECHNICAL PROFICIENCY: Valid historical control data ranges of the controls have been provided over the period of November 2013 to November 2016.

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 Positive control Positive control
3-minute treatment(OD570) 1-hour treatment(OD570) 3-minute treatment(OD570) 1-hour treatment(OD570) 3-minute treatment(% viability) 1-hour treatment(% viability)
Range 1.324 – 2.615 1.361 – 2.352 0.0172 – 0.56 0.046 – 0.339 6 – 25 3 – 13
Mean 1.84 1.85 0.19 0.14 11.03 7.45
SD 0.26 0.22 0.09 0.06 4.39 2.51
n 81 83 80 77 38 38
Interpretation of results:
other: Not corrosive
Remarks:
Based on CLP criteria (Annex I 1272/2008/EC)
Conclusions:
Based on the results obtained, it can be concluded that Cedarwood Virginia oil is not corrosive to skin and does not need to be classified accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
Executive summary:

The skin corrosion potential of Cedarwood Virginia oil was tested according to OECDTG431. A human three dimensional epidermal model (EpiDerm) was exposed topically to 50µL undiluted Cedarwood Virginia oil, Milli-Q (negative control), or Potassium hydroxide (positive control) for 3 minutes, or 1 hour. Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 94% and 103%, respectively. Both the negative and the positive control were considered valid. The mean relative tissue viability for Cedarwood Virginia oil was above 50% after the 3-minute treatment and above 15% after the 1-hour treatment. Based these results, the test substance does not need to be classified for skin corrosion in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
The full read across justification report is attached under "Attached justification".

20 December 2017 READ-ACROSS STUDY / CW TX OIL TERPENES (CEDRENE) / SKIN IRRITATION I&B9W8768R001F2.0

According to Annex VII, 8.1 of the REACh Regulation (EC) No 1907/2006, Skin irritation is standard information required for the registration of substances manufactured or imported in quantities of one tonne per year or more. However, according to Annex XI, 1.5 of the REACH Regulation, Read-across and grouping approaches can be used to adapt the standard testing regime. This read-across study report follows notably the recommendations made by the European Chemicals Agency in its “Guidance on information requirements and chemical safety assessment Chapter R.6 – QSARs and grouping of chemicals” (ECHA, 2008) and in its document “Read-Across Assessment Framework (RAAF)” (ECHA, 2017).

A read-across approach appears appropriate to predict the endpoint “Skin irritation” for the substance Cedarwood Texas oil distilled - Terpenes 1 (Cedrene) [CW TX T1] because:

A skin irritation study and a skin corrosion study, according to OECD test guidelines 439 and 431, respectively, are available for the substance Cedarwood Virginia oil [CW Virginia], which composition is very similar and in which Cedrol and Cedrene-alpha were identified as the constituents driving the skin irritation effect.

Analysis of literature and data available for the constituent thujopsene tends to confirm that variations in this constituent, which is the main difference with the source and target substances, do not significantly influence the skin irritation;

This report follows the RAAF method and so presents:
1) The hypothesis: analogue read-across approach, based on the similarity of the structures and the potential skin irritation effects for these types of structures;
2) The scientific justifications (“Assessment Elements”) and their evaluation (“Assessment Options”); which demonstrate the confidence that can be put in this prediction.
3) The conclusions, usable for classification assessment or risk assessment, which are summarised hereafter.
Reason / purpose for cross-reference:
read-across source
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 1002960562
- Expiration date of the lot/batch: 31 August 2017

STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none, the liquid test item was applied undiluted (50 µl) directly on top of the tissue.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minute exposure
Value:
94
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 hour exposure
Value:
103
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: yes, the non-specific reduction of MTT by the test item was -0.46% and 0.29% of the negative control tissues after 3 minutes and 1 hour respectively.
- Colour interference with MTT: yes

DEMONSTRATION OF TECHNICAL PROFICIENCY: Valid historical control data ranges of the controls have been provided over the period of November 2013 to November 2016.

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 Positive control Positive control
3-minute treatment(OD570) 1-hour treatment(OD570) 3-minute treatment(OD570) 1-hour treatment(OD570) 3-minute treatment(% viability) 1-hour treatment(% viability)
Range 1.324 – 2.615 1.361 – 2.352 0.0172 – 0.56 0.046 – 0.339 6 – 25 3 – 13
Mean 1.84 1.85 0.19 0.14 11.03 7.45
SD 0.26 0.22 0.09 0.06 4.39 2.51
n 81 83 80 77 38 38
Interpretation of results:
other: Not corrosive
Remarks:
Based on CLP criteria (Annex I 1272/2008/EC)
Conclusions:
The skin corrosion potential of Cedarwood Texas Terpenes (cedrene) was evaluated by read across with the source study for Cedarwood Virginia oil. Based on the results obtained, it can be concluded that Cedarwood Texas Terpenes (cedrene) is not corrosive to skin and does not need to be classified accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
Executive summary:

The skin corrosion potential of Cedarwood Texas Terpenes (cedrene) was evaluated by read across with the source study for Cedarwood Virginia oil. Cedarwood Virginia oil was tested according to OECDTG431. A human three dimensional epidermal model (EpiDerm) was exposed topically to 50µL undiluted Cedarwood Virginia oil, Milli-Q (negative control), or Potassium hydroxide (positive control) for 3 minutes, or 1 hour. Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 94% and 103%, respectively. Both the negative and the positive control were considered valid. The mean relative tissue viability for Cedarwood Virginia oil was above 50% after the 3-minute treatment and above 15% after the 1-hour treatment. Based these results, the test substance does not need to be classified for skin corrosion in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 Mar 2017 - 10 Apr 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)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
GLP compliance:
yes
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: B-64543
- Expiration date of the lot/batch: 25 January 2019

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

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

OTHER SPECIFICS: UVCB
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Adult donors
Source strain:
other: 16-08k008XP1-001 + 16-08k008XP2-001 + 10-kera-005 + 08-KERA-001
Details on animal used as source of test system:
Not relevant
Justification for test system used:
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise 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, Batch no.: 17-EKIN-013)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37.0 ± 1.0°C (actual range 35.4 - 36.9°C), with the exception of the test item incubation for 15 minutes at room temperature
- Temperature of post-treatment incubation (if applicable): 37.0 ± 1.0°C (actual range 35.4 - 36.9°C)

REMOVAL OF TEST MATERIAL AND CONTROLS
- Washing steps: 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: 0.3 mg/ml- Incubation time: 3 hours at 37°C
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the relative mean tissue viability of three viability tissues after 15 minutes exposure and 42 hours post incubation is less than or equal to 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
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 µl

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 25 µl PBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 µl SDS
- Concentration (if solution): 5% in PBS
Duration of treatment / exposure:
15 ± 0.5 minutes at room temperature
Duration of post-treatment incubation (if applicable):
42 hours at 37°C
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean (percentage of control)
Value:
97
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
Mean tissue viability (percentage of control): 3.7%
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
Terpenes 2 (Thujopsene), Cedarwood Texas oil distilled was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because no colour changes were observed it was concluded that Cedar atlantica oil did not interact with the MTT endpoint.

ACCEPTANCE OF RESULTS:

a) The absolute mean OD570 (optical density at 570 nm) of the three tissues of the negative control was within the laboratory historical control data range.
b) The mean relative tissue viability of the positive control was <50% (3.7%) relative to the negative control.
c) The SD calculated from individual % tissue viabilities of the three identically treated replicates was <18.
Interpretation of results:
other: Not Irritating
Remarks:
Based on CLP criteria (1272/2008/EC).
Conclusions:
After treatment with the test item Cedarwood Texas oil distilled - Terpenes (Thujopsene) , the relative cell viability values (compared to the negative control tissues) was 97%. This value is above the threshold for irritancy of 50%. Therefore it can be concluded that the test substance is not irritant to skin and does not need to be classified for skin irritation in accordance with the criteria outlined in Annex I of CLP (1272/2008/EC).
Executive summary:

Cedarwood Texas oil distilled - Terpenes (Thujopsene) distilled was evaluated for its ability to induce skin irritation according to OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method). The possible skin irritation potential of the test item was tested through topical application for 15 minutes. Terpenes 2 (Thujopsene), Cedarwood Texas oil distilled was applied undiluted (25 µl), directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed.  The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 97%. Since the mean relative tissue viability for the test item was above 50% after 15 ± 0.5 minutes treatment Terpenes 2 (Thujopsene), Cedarwood Texas oil distilled is considered to be non-irritant. The positive control and the negative control tissues functioned properly and the results were within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 6% for the negative and positive control, indicating that the test system functioned properly. In conclusion, Terpenes 2 (Thujopsene), Cedarwood Texas oil distilled is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report. 

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 Mar 2017 - 03 Apr 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: 1002960562
- Expiration date of the lot/batch: 31 August 2017
- Purity test date: 100.0%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: Stable under storage conditions until expiration date

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- The liquid test item was applied undiluted

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 17-EKIN-013) SkinEthic Laboratories, Lyon, France.
Source strain:
other: Human
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): Batch no.: 17-EKIN-013
- Production date: 28 Mar 2017
- Date of initiation of testing: 28 Mar 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 (actual range 35.9 - 37.0°C)

REMOVAL OF TEST MATERIAL AND CONTROLS
- Observable damage 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 13%, indicating that the test system functioned properly.

NUMBER OF REPLICATE TISSUES: triplicates

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues: In addition to the normal procedure, three killed tissues treated with test item and three killed non treated tissues were used for the cytotoxicity evaluation with MTT.
- Procedure used to prepare the killed tissues (if applicable): Living epidermis was transferred to 12 well plates and incubated with 2 ml Milli-Q for 48 ± 1 hours. After incubation, killed epidermis was stored at ≤ -15°C.
- N. of replicates: three
- Method of calculation used:

Nonspecific MTT reduction (NSMTT) was calculated as the difference between the OD of untreated killed tissues (ODku) and test item treated killed tissues (ODkt) expressed as percentage of the mean of the negative control tissues (ODnc).
NSMTT = [(ODkt – ODku)/ODnc] * 100

True MTT metabolic conversion (TODtt) is the difference between the OD of test item treated viable tissues (ODtv) and the difference between ODkt and ODku.
TODtt = [ODtv – (ODkt-ODku)]

The relative viability of these tissues is the true MTT metabolic conversion (TODtt) as a percentage of the mean of the negative control tissues. (In case the %NSMTT ≤ 0.0, there is no need to calculate the TODtt.)
% viability = [TODtt/ODnc] * 100

Non-Specific Color in Killed Tissues (NSCkilled) are calculated using the formula below
%NSCkilled = (OD (killed tissues without MTT) / OD negative control) x 100
OD = OD colored tissue (MTT assay) – OD colored living tissue (no MTT assay) – TODtt + OD (killed tissues without MTT)
Viability = (OD / OD negative control) * 100.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 3

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- 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:
41
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: not reported
- Direct-MTT reduction: yes


DEMONSTRATION OF TECHNICAL PROFICIENCY:
The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 3.7%. 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 13%, indicating that Cedarwood Oil Virginia 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=0.16, 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: Irritant Cat. 2
Remarks:
Based on CLP criteria (Annex I 1272/2008/EC)
Conclusions:
Under the conditions of this test, the relative mean tissue viability for the test item determined to be 41%. This value is below the threshold for irritancy of ≤50%. Based on the results obtained, it can be concluded that Cedarwood Virginia Oil is at least an irritant to skin, in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
Executive summary:

The skin irritation potential of Cedarwood Virginia Oil was tested in accordance to OECDTG439. Undiluted Cedarwood Virginia 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. As Cedarwood Virginia Oil interacted with the MTT, three killed tissues treated with test item and three killed untreated tissues were also used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by Cedarwood Oil Virginia was -0.06% of the negative control tissues.

 The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with Cedarwood Oil Virginia compared to the negative control tissues was 41%. Since the mean relative tissue viability for Cedarwood Oil Virginia was below 50% after 15 ± 0.5 minutes treatment it is considered to be an irritant. Both the positive 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 13%, indicating that the test system functioned properly.

 In conclusion, Cedarwood Oil Virginia is an irritant in the in vitro skin irritation test under the experimental conditions described in the report. (1272/2008/EC).

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
The full read across justification report is attached under "Attached justification".

20 December 2017 READ-ACROSS STUDY / CW TX OIL TERPENES (CEDRENE) / SKIN IRRITATION I&B9W8768R001F2.0

According to Annex VII, 8.1 of the REACh Regulation (EC) No 1907/2006, Skin irritation is standard information required for the registration of substances manufactured or imported in quantities of one tonne per year or more. However, according to Annex XI, 1.5 of the REACH Regulation, Read-across and grouping approaches can be used to adapt the standard testing regime. This read-across study report follows notably the recommendations made by the European Chemicals Agency in its “Guidance on information requirements and chemical safety assessment Chapter R.6 – QSARs and grouping of chemicals” (ECHA, 2008) and in its document “Read-Across Assessment Framework (RAAF)” (ECHA, 2017).

A read-across approach appears appropriate to predict the endpoint “Skin irritation” for the substance Cedarwood Texas oil distilled - Terpenes 1 (Cedrene) [CW TX T1] because:

A skin irritation study and a skin corrosion study, according to OECD test guidelines 439 and 431, respectively, are available for the substance Cedarwood Virginia oil [CW Virginia], which composition is very similar and in which Cedrol and Cedrene-alpha were identified as the constituents driving the skin irritation effect.

Analysis of literature and data available for the constituent thujopsene tends to confirm that variations in this constituent, which is the main difference with the source and target substances, do not significantly influence the skin irritation;

This report follows the RAAF method and so presents:
1) The hypothesis: analogue read-across approach, based on the similarity of the structures and the potential skin irritation effects for these types of structures;
2) The scientific justifications (“Assessment Elements”) and their evaluation (“Assessment Options”); which demonstrate the confidence that can be put in this prediction.
3) The conclusions, usable for classification assessment or risk assessment, which are summarised hereafter.
Reason / purpose for cross-reference:
read-across source
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: 1002960562
- Expiration date of the lot/batch: 31 August 2017
- Purity test date: 100.0%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: Stable under storage conditions until expiration date

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- The liquid test item was applied undiluted

Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Main experiment in triplicate
Value:
41
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: not reported
- Direct-MTT reduction: yes


DEMONSTRATION OF TECHNICAL PROFICIENCY:
The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 3.7%. 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 13%, indicating that Cedarwood Oil Virginia 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=0.16, 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: Irritant Cat 2
Remarks:
Based on CLP criteria (Annex I 1272/2008/EC)
Conclusions:
The skin irritation potential of Cedarwood Texas Terpenes (cedrene) was evaluated by read across with the source study for Cedarwood Virginia oil. Based on the results obtained, it can be concluded that Cedarwood Texas Terpenes (cedrene) is at least an irritant to skin.
Executive summary:

The skin irritation potential of Cedarwood Texas Terpenes (cedrene) was evaluated by read across with the source study for Cedarwood Virginia oil.

The skin irritation potential of Cedarwood Virginia Oil was tested in accordance to OECDTG439. Undiluted Cedarwood Virginia 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. As Cedarwood Virginia Oil interacted with the MTT, three killed tissues treated with test item and three killed untreated tissues were also used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by Cedarwood Oil Virginia was -0.06% of the negative control tissues.

 The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with Cedarwood Oil Virginia compared to the negative control tissues was 41%. Since the mean relative tissue viability for Cedarwood Oil Virginia was below 50% after 15 ± 0.5 minutes treatment it is considered to be an irritant. Both the positive 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 13%, indicating that the test system functioned properly.

 In conclusion, Cedarwood Oil Virginia is an irritant in the in vitro skin irritation test under the experimental conditions described in the report.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 Apr 2017 - 04 Apr 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:
July 26, 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: B-64543
- Expiration date of the lot/batch: 25 January 2019

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

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, 's Hertogenbosch, The Netherlands
- 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. 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-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)).
- Time interval prior to initiating testing: Bovine eyes were used as soon as possible after slaughter
- indication of any existing defects or lesions in ocular tissue samples: no
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 μl)
- Concentration (if solution): pure

Duration of treatment / exposure:
10 +/- 1 minutes
Duration of post- treatment incubation (in vitro):
120 +/- 10 minutes
Number of animals or in vitro replicates:
triplicates
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 and 1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder). 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 prior to the start of the exposure

QUALITY CHECK OF THE ISOLATED CORNEAS
Opacity determinations were performed on each of the received 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
three

NEGATIVE CONTROL USED
Yes, untreated

POSITIVE CONTROL USED
Yes, Ethanol

APPLICATION DOSE AND EXPOSURE TIME
Dose: pure 100%
Exposure time: 10 minutes

TREATMENT METHOD: closed chamber

POST-EXPOSURE INCUBATION PERIOD: yes 120 +/- 10 minutes

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least 2 (once with MEM with phenol red (Eagle’s Minimum Essential Medium) and thereafter with cMEM)

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity:

The opacity of a cornea was measured by measuring the diminution of light passing through the cornea. The light is measured as illuminance (I = luminous flux per area, unit: lux) by a light meter.
The opacity value (measured with the device OP-KIT) is calculated according to:
Opacity= (empirically determined illuminance through a cornea holder, devided by the measured illuminance through a holder with cornea - 0,9894)/0,0251
The change in opacity for each individual cornea (including the negative control) is calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity of each cornea treated with the test item or positive control is calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each test item or positive control treated cornea. The mean opacity value of each treatment group is calculated by averaging the corrected opacity values of the treated corneas for each treatment group.

- Corneal permeability: passage of sodium fluorescein dye measured with the aid of a microtiter plate reader (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used.

The assay is considered acceptable if:
- The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean.
- The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.

In vitro score ranges used:
≤ 3 No Category
> 3; ≤ 55 No prediction can be made
>55 Category 1
Irritation parameter:
in vitro irritation score
Run / experiment:
Main (triplicate)
Value:
-1 - 0.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
-0.3 - 0.5
Positive controls validity:
valid
Remarks:
45.2 - 61.5
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: none

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 51 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
Negative control Permeability -0.016 – 0.042
Negative control In vitro Irritancy Score -2.8 – 3.0
Positive control In vitro Irritancy Score 34.7 – 78.2
Interpretation of results:
other: Not irritating
Remarks:
Based on CLP (1272/2008/EC).
Conclusions:
Based on the results obtained in the presented study, Cedarwood Texas oil distilled - Terpenes (Thujopsene) does not need to be classified for eye irritation in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
Executive summary:

The eye hazard potential of Cedarwood Texas oil distilled - Terpenes (Thujopsene) was determined by measuring its ability to induce opacity and increase permeability according to OECDTG 437. The eye damage of the test item was tested through topical application for 10 minutes. The test item was applied as it is (750 μl) directly on top of the corneas. 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 51 and 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. The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 0.0 after 10 minutes of treatment. In conclusion, since Cedarwood Texas oil distilled - Terpenes (Thujopsene) induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage, in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 Apr 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:
July 26, 2013
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- batch No.of test material: 1002960562 provided by sponsor
- Expiration date of the lot/batch: 31 August 2017
- Purity test date: 22 August 2017

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.
Species:
cattle
Strain:
not specified
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.
Vehicle:
unchanged (no vehicle)
Controls:
yes
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): The medium from the anterior compartment was removed and 750 µl of either the negative control, positive control (Ethanol) or test item was introduced onto the epithelium of the cornea.
- 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:
triplicates
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-glutamine (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 (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:
Identification Ethanol
Identification number RS532
CAS Number 64-17-5
Molecular formula C2H5OH
Molecular weight 46.07
Appearance Clear colourless liquid
Batch K47177483
Purity >99.9%
Storage conditions At room temperature
Stable under storage conditions until 31 October 2020

APPLICATION DOSE AND EXPOSURE TIME: Undiluted, 10 minutes

TREATMENT METHOD: 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.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period:
- POST-EXPOSURE INCUBATION: yes; 120 +/- 10 minutes

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)

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used.
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.
INTERPRETATION
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 given hereafter:
≤ 3 No Category
> 3; ≤ 55 No prediction can be made
>55 Category 1
Irritation parameter:
in vitro irritation score
Run / experiment:
1
Value:
>= 0.6 - <= 2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / 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 51 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.

Treatment

Mean

Opacity

Mean

Permeability

MeanIn vitroIrritation Score

Negative control

0.1

0.007

0.2

Positive control

(Ethanol)

19.8

2.056

50.6

Test item

1.0

0.024

1.4

Interpretation of results:
other: not classified
Remarks:
based on CLP criteria (Annex I of 1272/2008/EC)
Conclusions:
Cedarwood Oil Virginia 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 irritation potential of Cedarwood Oil Virginia 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.

Cedarwood Oil Virginia was applied as received to the test system, and did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 1.4 after 10 minutes of treatment. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical rangeindicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (Ethanol) was 51 and 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.  In conclusion, since Cedarwood Oil Virginia induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage according to the classification criteria outlined in Annex I of 1272/2008/EC (CLP).

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
The full read across justification report is attached under "Attached justification".

20 December 2017 READ-ACROSS STUDY / CW TX OIL TERPENES I&B9W8768R001F0.2

According to Annex VII, 8.2 of the REACh Regulation (EC) No 1907/2006, Eye irritation is standard information required for the registration of substances manufactured or imported in quantities of one tonne per year or more. However, according to Annex XI, 1.5 of the REACH Regulation, Read-across and grouping approaches can be used to adapt the standard testing regime. This read-across study report follows notably the recommendations made by the European Chemicals Agency in its “Guidance on information requirements and chemical safety assessment Chapter R.6 – QSARs and grouping of chemicals” (ECHA, 2008) and in its document “Read-Across Assessment Framework (RAAF)” (ECHA, 2017).

A read-across approach appears appropriate to predict the endpoint “Eye irritation” for the substance Cedarwood Texas oil distilled - Terpenes 1 (Cedrene) [CW TX T1] because:

An eye irritation study, according to OECD test guideline 437, is available for the substance Cedarwood Virginia oil, CW Virginia oil [CW Virginia], which composition is very similar and in which Cedrene-alpha, which may be the only constituent with possible eye irritation effects, is in similar or higher concentration.

Analysis of the literature and of available data for the constituent thujopsene tends to confirm that the variation in this constituent, which is the main difference with the source substance, is not expected to significantly influence the eye irritation potential.

This report follows the RAAF method and so presents:
1) The hypothesis: analogue read-across approach, based on the similarity of the structures and the potential for eye irritation effects for these types of structures;
2) The scientific justifications (“Assessment Elements”) and their evaluation (“Assessment Options”); which demonstrate the confidence that can be put in this prediction.
3) The conclusions, usable for classification assessment or risk assessment, which are summarised hereafter.
Reason / purpose for cross-reference:
read-across source
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- batch No.of test material: 1002960562 provided by sponsor
- Expiration date of the lot/batch: 31 August 2017
- Purity test date: 22 August 2017

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.
Controls:
yes
yes, concurrent positive control
yes, concurrent negative control
Irritation parameter:
in vitro irritation score
Run / experiment:
1
Value:
>= 0.6 - <= 2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / 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 51 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.

Treatment

Mean

Opacity

Mean

Permeability

MeanIn vitroIrritation Score

Negative control

0.1

0.007

0.2

Positive control

(Ethanol)

19.8

2.056

50.6

Test item

1.0

0.024

1.4

Interpretation of results:
other: not classified
Remarks:
based on CLP criteria (Annex I of 1272/2008/EC)
Conclusions:
The eye irritation potential of Cedarwood Texas Terpenes (cedrene) was evaluated by read across with the source study for Cedarwood Virginia oil. Based on these results, Cedarwood Texas Terpenes (cedrene) 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 irritation potential of Cedarwood Texas Terpenes (cedrene) was evaluated by read across with the source study for Cedarwood Virginia oil. The eye irritation potential of Cedarwood Oil Virginia 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.

Cedarwood Oil Virginia was applied as received to the test system, and did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 1.4 after 10 minutes of treatment. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical rangeindicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (Ethanol) was 51 and 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.  In conclusion, since Cedarwood Oil Virginia induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage 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/corrosion

The skin irritation/corrosion of Cedarwood Texas oil distilled - Terpenes was assessed. Cedarwood Texas oil distilled - Terpenes (Thujopsene) was tested in an OECD TG 439 study, of which the summaries are presented below. The skin irritation/corrosion of Cedarwood Texas oil distilled - Terpenes (Cedrene) was assessed by using read across from Cedarwood Virginia oil. The summary of the read-across study is presented below, and the read across justification is attached to the oral toxicity target record.

Cedarwood Texas oil distilled - Terpenes (Thujopsene): Skin irritation (OECDTG439)

Cedarwood Texas oil distilled - Terpenes (Thujopsene) distilled was evaluated for its ability to induce skin irritation according to OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method). The possible skin irritation potential of the test item was tested through topical application for 15 minutes. Terpenes 2 (Thujopsene), Cedarwood Texas oil distilled was applied undiluted (25 µl), directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed.  The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 97%. Since the mean relative tissue viability for the test item was above 50% after 15 ± 0.5 minutes treatment Terpenes 2 (Thujopsene), Cedarwood Texas oil distilled is considered to be non-irritant. The positive control and the negative control tissues functioned properly and the results were within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 6% for the negative and positive control, indicating that the test system functioned properly. In conclusion, Terpenes 2 (Thujopsene), Cedarwood Texas oil distilled is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report. 

Read-across source substance Cedarwood Virginia oil: Skin irritation (OECDTG439)

The skin irritation potential of Cedarwood Virginia oil was tested in accordance to OECDTG439. Undiluted Cedarwood Virginia 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. As Cedarwood Virginia oil interacted with the MTT, three killed tissues treated with test item and three killed untreated tissues were also used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by Cedarwood Virginia oil was -0.06% of the negative control tissues.

 The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with Cedarwood Virginia oil compared to the negative control tissues was 41%. Since the mean relative tissue viability for Cedarwood Oil Virginia was below 50% after 15 ± 0.5 minutes treatment it is considered to be an irritant. Both the positive 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 13%, indicating that the test system functioned properly.

 In conclusion, Cedarwood Virginia oil is an irritant in the in vitro skin irritation test under the experimental conditions described in the report. (1272/2008/EC).

Read-across source substance Cedarwood Virginia oil: Skin corrosion (OECDTG431)

The skin corrosion potential of Cedarwood Virginia oil was tested according to OECDTG431. A human three dimensional epidermal model (EpiDerm) was exposed topically to 50µL undiluted

Cedarwood Virginia oil, Milli-Q (negative control), or Potassium hydroxide (positive control) for 3 minutes, or 1 hour. Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 94% and 103%, respectively. Both the negative and the positive control were considered valid. The mean relative tissue viability for Cedarwood Virginia oil was above 50% after the 3-minute treatment and above 15% after the 1-hour treatment. Based these results, the test substance does not need to be classified for skin corrosion in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).

Eye irritation

The eye irritation of Cedarwood Texas oil distilled - Terpenes was assessed. Cedarwood Texas oil distilled - Terpenes (Thujopsene) was tested in an OECD437 study, of which the summaries are presented below. The eye irritation of Cedarwood Texas oil distilled - Terpenes (Cedrene) was assessed by using read across from Cedarwood Virginia oil. The summary of the read-across study is presented below, and the read across justification is attached to the oral toxicity target record.

Cedarwood Texas oil distilled - Terpenes (Thujopsene): Eye irritation (OECDTG437)

The eye hazard potential of Cedarwood Texas oil distilled - Terpenes (Thujopsene) was determined by measuring its ability to induce opacity and increase permeability according to OECDTG 437. The eye damage of the test item was tested through topical application for 10 minutes. The test item was applied as it is (750 μL) directly on top of the corneas. 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 51 and 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. The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 0.0 after 10 minutes of treatment. In conclusion, since Cedarwood Texas oil distilled - Terpenes (Thujopsene) induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage, in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).

Read-across source substance Cedarwood Virginia oil: Eye irritation (OECDTG437)

The eye irritation potential of Cedarwood Oil Virginia 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.

Cedarwood Oil Virginia was applied as received to the test system, and did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 1.4 after 10 minutes of treatment. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical rangeindicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (Ethanol) was 51 and 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.  In conclusion, since Cedarwood Oil Virginia induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage according to the classification criteria outlined in Annex I of 1272/2008/EC (CLP).

Justification for classification or non-classification

Based on the available data it can be concluded that Cedarwood Texas oil distilled - Terpenes (Thujopsene) is not irritant to skin or eye and does not need to be classified for skin or eye irritation in accordance with the criteria outlined in Annex I of CLP (1272/2008/EC).

Based on the available read-across data Cedarwood Texas oil distilled - Terpenes (Cedrene) was determined to be a skin irritant but did not cause corrosion. Therefore the substance should be classified as skin irritant (Category 2) in accordance with the criteria outlined in Annex I of CLP (1272/2008/EC). Furthermore, no classification is required for eye irritation or serious eye damage in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).