Registration Dossier

Administrative data

Description of key information

Skin irritation in vitro using read across from Cedarwood Texas oil distilled (OECD TG 439): irritating

Skin corrosion in vitro using read across from Cedarwood Texas oil distilled (OECD TG 431): not corrosive

Eye irritation in vitro using read across from Cedarwood Texas oil distilled (OECD TG 438): not irritating

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:
20-07-2017 to 21-07-2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
adopted 29 July 2016
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
- Identification: Cedrol, Cedarwood Texas oil distilled
- Appearance: Light yellow, solid mass
- Batch: B-64530
- Purity/Composition: UVCB
- Test item storage: At room temperature
- Stable under storage conditions until: 25 January 2019 (expiry date)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test item should be equilibrated to 100°C for several minutes until completely liquefied to obtain a homogeneous sample.
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: MatTek Corporation
Source strain:
other: Keratinocyte strain 00267
Justification for test system used:
Recommended test system in international guidelines (OECD and EC).
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model
- Tissue batch number(s): Lot no.: 26708 Kit L and Kit M
- Production date: 19-07-2017
- Date of initiation of testing: 20-07-2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 min room temperature, 1 hour 37ºC

REMOVAL OF TEST MATERIAL AND CONTROLS
- Washing step: washed with phosphate buffered saline
- Damage: no visual damage

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT concentrate (5 mg/ml) diluted (1:5) with MTT diluent (supplemented DMEM)
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The absolute mean OD570 of the two tissues of the negative control should reasonably be within the laboratory historical control data range. The mean relative tissue viability following 1-hour exposure to the positive control should be <15 %.
- Reproducibility: In the range 20 - 100% viability, the Coefficient of Variation (CV) between tissue replicates should be  30%.

NUMBER OF REPLICATE TISSUES: 2

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:
- 1 experiment with 3 minute application (in duplicate)
- 1 experiment with 1 hour application (in duplicate)

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin 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%.
- The test substance is considered to be non-corrosive to skin 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

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50µL
- Concentration (if solution):8N
Duration of treatment / exposure:
3 min / 1 hour
Number of replicates:
2
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 min exposure
Value:
104
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:
116
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: no

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative/positive control:
The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit <=2.8) and the laboratory historical control data range.
- Acceptance criteria met for variability between replicate measurements: The mean relative tissue viability following the 1-hour exposure to the positive control was 10%.
In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was <=8.0%, indicating that the test system functioned properly.
- Range of historical values (OD570):

Negative control Positive control Positive control
3-minute treatment 1-hour treatment 3-minute treatment 1-hour treatment 3-minute treatment 1-hour treatment
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 skin corrosive
Remarks:
in accordance with EU CLP (EC 1272/2008 and its updates)
Conclusions:
Under the conditions of the test, the Cedrol, Cedarwood Texas oil distilled was not corrosive.
Executive summary:

Cedrol, Cedarwood Texas oil distilled was evaluated for its ability to induce skin corrosion on a human three dimensional epidermal model according to OECD TG 431.  Cedrol, Cedarwood Texas oil distilled was applied topically for 3 minutes and 1 hour. The test substance was equilibrated to 100°C for several minutes until completely liquefied to obtain a homogeneous liquid sample.  Cedrol, Cedarwood Texas oil distilled (50 µl) was applied directly on top of the skin tissue.  

The positive control had a mean relative tissue viability of 10% after the 1-hour exposure.  The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥ 0.8 and upper acceptance limit ≤ 2.8) and the laboratory historical control data range.  In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was ≤ 8.0%, indicating that the test system functioned properly.

The relative mean tissue viability obtained after 3-minute and 1-hour treatments with Cedrol, Cedarwood Texas oil distilled compared to the negative control tissues was 104% and 116%, respectively.  Because the mean relative tissue viability for Cedrol, Cedarwood Texas oil distilled was above 50% after the 3-minute treatment and above 15% after the 1-hour treatment Cedrol, Cedarwood Texas oil distilled is considered to be not corrosive.

In conclusion, Cedrol, Cedarwood Texas oil distilled is not corrosive in the in vitro skin corrosion test under the experimental conditions described.

Endpoint:
skin corrosion: 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:
read-across source
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 min exposure
Value:
104
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:
116
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Interpretation of results:
other: not skin corrosive
Remarks:
in accordance with EU CLP (EC 1272/2008 and its updates)
Conclusions:
Based on the results of the study for read-across substance Cedarwood Texas oil distilled, Cedarwood oil Himalayan is considered not corrosive.
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 Apr 2017 - 24 Apr 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
28 July 2015
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
GLP compliance:
yes (incl. certificate)
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-64530
- Expiration date of the lot/batch: 25 January 2019
- Purity test date: 26 January 2016

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: Test item was equilibrated to 100°C for several minutes until completely liquefied to obtain a homogeneous sample.

OTHER SPECIFICS: UVCB
Test system:
artificial membrane barrier model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 17-EKIN-016)
Source strain:
other: 09-KERA-002 and 11-KERA-002
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-016
- Production date: 19 April 2017
- Date of initiation of testing: 19 April 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 36.2 – 37.4°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 14%, indicating that the test system functioned properly.

NUMBER OF REPLICATE TISSUES: triplicates

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

DECISION CRITERIA
- The test substance is considered to be irritant to skin if the relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
- The test substance is considered to be non-irritant to skin if the relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 25µL undiluted

NEGATIVE CONTROL
- Amount(s) applied: 25µL undiluted

POSITIVE CONTROL
- Amount(s) applied: 25µL
- Concentration (if solution): 5%
Duration of treatment / exposure:
15 ± 0.5 minutes
Duration of post-treatment incubation (if applicable):
42 hours at 37°C
Number of replicates:
three
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Main experiment in triplicate
Value:
17
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: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 8.8%. 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 14%, indicating that the test system functioned properly.

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

Negative control: Absorption OD570=0.676-1.336, Mean=1.01, SD=0.016, n=155
Positive control: Absorption OD570=0.036-0.549, Mean=0.16, SD=0.10, n=154
Positive control: Viability %= 2.85-45.43, Mean=15.74, SD=9.22, n=163
Interpretation of results:
other: Skin Irrit. 2
Remarks:
in accordance with EU CLP (EC 1272/2008 and its updates)
Conclusions:
Based on the results obtained, it can be concluded that Cedrol, Cedarwood Texas oil distilled is a skin irritant.
Executive summary:

The skin irritation potential of Cedrol, Cedarwood Texas oil distilled was tested in accordance to OECDTG439. The undiluted test substance was topically applied to EPISKIN-SMTM for 15 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed using MTT conversion measurements.

The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with  Cedrol, Cedarwood Texas oil distilled compared to the negative control tissues was 17%. Since the mean relative tissue viability for Cedrol, Cedarwood Texas oil distilled 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 14%, indicating that the test system functioned properly.

 In conclusion, Cedrol, Cedarwood Texas oil distilled was determined to be an 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:
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:
read-across source
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Main experiment in triplicate
Value:
17
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Interpretation of results:
other: Skin Irrit. 2
Remarks:
in accordance with EU CLP (EC 1272/2008 and its updates)
Conclusions:
Based on the results of the study for read-across substance Cedarwood Texas oil distilled, Cedarwood oil Himalayan is considered a skin irritant.
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:
11 Apr 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
26 July, 2013
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and batch No.of test material: Provided by sponsor, B-64530
- Expiration date of the lot/batch: 25 January 2019
- Purity test date: 26 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: Test item was equilibrated to 100°C for several minutes until completely liquefied to obtain a homogeneous sample.
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. Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µl of either the negative control, positive control or undiluted test item was introduced onto the epithelium of the cornea.
Duration of treatment / exposure:
Corneas were incubated in a horizontal position for 10 +/- 1 minutes at 32 +/- 1*C. After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Earle’s Minimum Essential Medium, Life Technologies) and thereafter with cMEM.
Duration of post- treatment incubation (in vitro):
Corneas were incubated for 120 +/- 10 minutes at 32 +/- 1*C with cMEM.
Number of animals or in vitro replicates:
Triplicate
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
"-The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded. The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium containing 1% (v/v) L-glutamine and 1% (v/v) Foetal Bovine Serum ). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 +/- 1*C. The corneas were incubated for the minimum of 1 hour at 32 +/- 1*C.

QUALITY CHECK OF THE ISOLATED CORNEAS:
-Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED:
- A negative control, physiological saline (Eurovet Animal Health, Bladel, The Netherlands) was included to detect non-specific changes in the test system and to provide a baseline for the assay endpoints.

POSITIVE CONTROL USED:
- Ethanol, Batch K47177483, Identification number RS532, Purity >99.9%
Stable under storage conditions until 31 October 2020

APPLICATION DOSE AND EXPOSURE TIME:
- Undiluted, 10 minutes

TREATMENT METHOD:
- The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 +/- 1*C. The corneas were incubated for the minimum of 1 hour at 32 +/- 1*C.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 2
- POST-EXPOSURE INCUBATION: yes; 120 +/- 10 minutes

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

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

ACCEPTABILITY CRITERIA
The assay is considered acceptable if:
1) The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean.
2) The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

EVALUATION CRITERIA
The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score: In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value) Additionally the opacity and permeability values were evaluated independently to determine whether the test item induced irritation through only one of the two endpoints. The IVIS cut-off values for identifying the test items as inducing serious eye damage (UN GHS Category 1) and test items not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are: ≤ 3: No Category, > 3 ≤ 55: No prediction can be made, >55: Category 1
Irritation parameter:
in vitro irritation score
Run / experiment:
Main
Value:
>= -0.6 - <= 0.1
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: not reported

DEMONSTRATION OF TECHNICAL PROFICIENCY:
- The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (Ethanol) was 61 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

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

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

Treatment

Mean

Opacity

Mean

Permeability

MeanIn vitroIrritation Score

Negative control

-0.5

 0.003

-0.4

Positive control

(Ethanol)

19.6

2.737

60.7

Test item

-0.2

0.059

0.7

Interpretation of results:
other: not eye irritating
Remarks:
in accordance with EU CLP (EC 1272/2008 and its updates)
Conclusions:
Cedrol, Cedarwood Texas oil distilled induced an IVIS ≤ 3. Based on these results, the test substance is not considered to be an eye irritant.
Executive summary:

The eye hazard potential of Cedrol, Cedarwood Texas oil distilled was evaluated according to OECD Guideline 437 (BCOP test). The eye damage was assessed through topical application of 750 µl of the undiluted Cedrol, Cedarwood Texas oil distilled for 10 minutes on top of the corneas. Both the negative control and the positive control (Ethanol) were considered valid. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. Cedrol, Cedarwood Texas oil distilled did not induce ocular irritation (no opacity and no permeability), resulting in a mean in vitro irritancy score of 0.7 after 10 minutes of treatment. In conclusion, since Cedrol, Cedarwood Texas oil distilled induced an IVIS ≤ 3, and the test substance is not considered to be an eye irritant.

Endpoint:
eye 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:
read-across source
Irritation parameter:
in vitro irritation score
Run / experiment:
Main
Value:
>= -0.6 - <= 0.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Interpretation of results:
other: not eye irritating
Remarks:
in accordance with EU CLP (EC 1272/2008 and its updates)
Conclusions:
Based on the results of the study for read-across substance Cedarwood Texas oil distilled, Cedarwood Topped China is not considered to be an eye irritant.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Additional information

The corrosive and irritation potential of Cedarwood oil Himalayan was assessed by using read across from Cedarwood Texas oil distilled. First the experimental information of Cedarwood Texas oil distilled will be summarised. Thereafter the read across justification is presented. The accompanying files are attached in the present endpoint summary.

Skin irritation

The skin irritation potential of Cedrol, Cedarwood Texas oil distilled was tested in accordance to OECDTG439. Undiluted test substance was topically applied to EPISKIN-SMTM for 15 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed using MTT conversion measurements.

The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with  Cedrol, Cedarwood Texas oil distilled compared to the negative control tissues was 17%. Since the mean relative tissue viability for Cedrol, Cedarwood Texas oil distilled 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 14%, indicating that the test system functioned properly.

 In conclusion, Cedrol, Cedarwood Texas oil distilled was determined to be an irritant in the in vitro skin irritation test under the experimental conditions described in this report.

Skin corrosion

Cedrol, Cedarwood Texas oil distilled was evaluated for its ability to induce skin corrosion on a human three dimensional epidermal model according to OECD TG 431.  Cedrol, Cedarwood Texas oil distilled was applied topically for 3 minutes and 1 hour. The test substance was equilibrated to 100°C for several minutes until completely liquefied to obtain a homogeneous liquid sample.  Cedrol, Cedarwood Texas oil distilled (50 µl) was applied directly on top of the skin tissue.   The positive control had a mean relative tissue viability of 10% after the 1-hour exposure.  The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥ 0.8 and upper acceptance limit ≤ 2.8) and the laboratory historical control data range.  In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was ≤ 8.0%, indicating that the test system functioned properly. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with Cedrol, Cedarwood Texas oil distilled compared to the negative control tissues was 104% and 116%, respectively.  Because the mean relative tissue viability for Cedrol, Cedarwood Texas oil distilled was above 50% after the 3-minute treatment and above 15% after the 1-hour treatment Cedrol, Cedarwood Texas oil distilled is considered to be not corrosive. In conclusion, Cedrol, Cedarwood Texas oil distilled is not corrosive in the in vitro skin corrosion test under the experimental conditions described.

Eye irritation

The eye hazard potential of Cedrol, Cedarwood Texas oil distilled was evaluated according to OECD Guideline 437 (BCOP test). The eye damage was assessed through topical application of the undiluted Cedrol, Cedarwood Texas oil distilled for 10 minutes on top of the corneas. Both the negative control and the positive control (Ethanol) were considered valid. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. Cedrol, Cedarwood Texas oil distilled did not induce ocular irritation (no opacity and no permeability), resulting in a mean in vitro irritancy score of 0.7 after 10 minutes of treatment. In conclusion, since Cedrol, Cedarwood Texas oil distilled induced an IVIS ≤ 3, therefore no classification is required for eye irritation or serious eye damage.

Read across justification

Mammalian toxicity and auto-ignition temperature of Essential oil of Cedarwood Himalayan obtained from the root of Cedrus deodara by distillation (CAS 91771-47-0; target) using read across from Cedarwood Texas oil distilled – Cedrol (CAS 91722-61-6; source)

Introduction and hypothesis for the analogue approach

Cedarwood Himalayan oil is a UVCB which consists of hydrocarbon constituents. Its major constituent is Cedrol. The full composition of Cedarwood Himalayan is presented in data matrix 1. For Cedarwood Himalayan oil no data are available for mammalian toxicity and auto flammability (see data matrix 2). 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 mammalian toxicity and the auto flammability of Cedarwood Himalayan oil, the mammalian toxicity data and auto flammability data of Cedarwood Texas oil distilled - Cedrol were used.The analogue substance-based read-across approach is selected because both the source and target cedarwood oils contain one major constituent, Cedrol. Sufficient reliable information is available for Cedarwood Texas oil distilled - Cedrol which can be used for read across.

 

Hypothesis:

Cedarwood Himalayan oil (target) is expected to have the same auto flammability as Cedarwood Texas oil distilled - Cedrol (source).

Cedarwood Himalayan oil (target) is expected to have the same mammalian toxicity as Cedarwood Texas oil distilled - Cedrol (source).

 

Available information:

 

Auto flammability:

The auto flammability of Texas Cedarwood oil, distilled was tested according to EU Method A.15 and DIN 51794. The test item Texas Cedarwood oil, distilled - Cedrol has an Auto flammability temperature of 260°C at 1016.6 hPa.

 

Acute oral toxicity:

The acute toxic potential of Cedarwood Texas oil distilled - Cedrol was assessed in an acute oral toxicity limit test performed similar to OECD TG 401. Ten rats were exposed to 5000 mg/kg bw Cedarwood Texas oil distilled - Cedrol via the oral route, and observed for clinical signs and mortality over an examination period of 14 days. Daily observations were performed. At the end of the study period no mortality was observed in any of the animals. Furthermore no symptoms occurred in the test animals.Based on these results the LD50 for acute oral toxicity was set at > 5000 mg/kg bw.

 

Skin irritation/corrosion:

Cedarwood Texas oil distilled - Cedrol was evaluated for its ability to induce skin corrosion on a human three dimensional epidermal model according to OECD TG 431.  Cedarwood Texas oil distilled - Cedrol was applied topically for 3 minutes and 1 hour. The test substance was equilibrated to 100°C for several minutes until completely liquefied to obtain a homogeneous liquid sample.  Cedarwood Texas oil distilled - Cedrol (50 µl) was applied directly on top of the skin tissue.  

The positive control had a mean relative tissue viability of 10% after the 1-hour exposure.  The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥ 0.8 and upper acceptance limit ≤ 2.8) and the laboratory historical control data range.  In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was ≤ 8.0%, indicating that the test system functioned properly.

The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test substance compared to the negative control tissues was 104% and 116%, respectively.  Because the mean relative tissue viability for Cedarwood Texas oil distilled - Cedrol was above 50% after the 3-minute treatment and above 15% after the 1-hour treatment –the test substance is considered to be not corrosive.

In conclusion, Cedarwood Texas oil distilled - Cedrol is not corrosive in the in vitro skin corrosion test under the experimental conditions described.

 

The skin irritation potential of Cedarwood Texas oil distilled - Cedrol was tested in accordance to OECDTG439. Undiluted Cedarwood Texas oil distilled - Cedrol was topically applied to EPISKIN-SMTM for 15 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed using MTT conversion measurements.

The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with – the test substance compared to the negative control tissues was 17%. Since the mean relative tissue viability 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 14%, indicating that the test system functioned properly.

 In conclusion, Cedarwood Texas oil distilled - Cedrol was determined to be an irritant in the in vitro skin irritation test under the experimental conditions described in this report.

 

Eye irritation:

The eye hazard potential of Cedarwood Texas oil distilled - Cedrol was evaluated according to OECD Guideline 437 (BCOP test). The eye damage was assessed through topical application of 750 µl of the undiluted substance for 10 minutes on top of the corneas. Both the negative control and the positive control (Ethanol) were considered valid. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. Cedarwood Texas oil distilled - Cedrol did not induce ocular irritation (no opacity and no permeability), resulting in a mean in vitro irritancy score of 0.7 after 10 minutes of treatment. In conclusion, since Cedarwood Texas oil distilled - Cedrol induced an IVIS ≤ 3, and the test substance is not considered to be an eye irritant.

 

Skinsensitisation:

The skin sensitisation potential of Cedarwood Texas oil distilled - Cedrol was tested according to OECD TG 429 (LLNA). Three experimental groups of five female CBA/J mice were treated with test item concentrations of 5, 10 or 25% w/w on three consecutive days, by open application on the ears. Test item concentrations selected for the main study were based on the results of a pre-screen test. Five vehicle control animals were similarly treated, but with the vehicle alone (AcOO).  No erythema was noted for any of the animals, Scaliness was noted on the ears of four animals treated at 25% between days 3 and 6. No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals. Body weights and body weight gain of experimental animals remained in the same range as controls over the main study period. Mean DPM/animal values for the experimental groups treated with test item concentrations 5, 10 and 25% were 561, 647 and 1524 DPM, respectively. The mean DPM/animal value for the vehicle control group was 285 DPM. The SI values calculated for the test item concentrations 5, 10 and 25% were 2.0, 2.3 and 5.4, respectively. The data showed a dose-response and an EC3 value (the estimated test item concentration that will give a SI =3) of 13.4% was calculated. Based on the results of this LLNA, Cedarwood Texas oil distilled - Cedrol is considered to be a skin sensitiser.

 

Mutagenicity:

The mutagenic potential of Cedarwood Texas oil distilled - Cedrol was evaluated according to OECD TG 471. In the dose-range finding test, the test item was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The test item precipitated on the plates at the dose level of 5000 μg/plate. Cytotoxicity was observed in tester strain TA100 at dose levels of 512 μg/plate and upwards in the absence and presence of S9-mix. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested.  Based on the results of the dose-range finding test, the test item was tested in the first mutation assay at a concentration range of 5.4 to 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. The test item precipitated on the plates at the dose level of 5000 μg/plate. Cytotoxicity was observed in all three tester strains in the absence and presence of S9-mix. In a follow-up experiment of the assay with additional parameters, the test item was tested at a concentration range of 5.4 to 5000 μg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test item was tested up to or beyond a precipitating dose level. Cytotoxicity was observed in all tester strains in the absence and presence of S9 -mix, except in tester strain WP2uvrA in the presence of S9-mix. Cedarwood Texas oil distilled - Cedrol did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment. In this study, acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly. In conclusion, based on the results of this study it is concluded that Cedarwood Texas oil distilled - Cedrol is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Target and Source chemical(s):The information on for Cedarwood Himalayan oil (target) and the information from Cedarwood Texas oil distilled - Cedrol (source) are presented in the data matrix 2 of this document. This includes physico-chemical properties and toxicological information, relevant for auto-flammability and mammalian toxicity.

 

Purity / Impurities:

The impurities are not relevant forCedarwood Himalayan oil(target) andCedarwood Texas oil distilled - Cedrol (source)as both substances are UVCBs (Natural Complex Substances).

Analogue approach justification

According to REACH Annex XI an analogue approach and structural alert information can be used to replace testing when information from different sources provides sufficient evidence to conclude that this substance has or does not have a particular dangerous property. The result derived should be applicable for C&L and/or risk assessment and be presented with adequate and reliable documentation.

Analogue selection:

Cedarwood Texas oil distilled - Cedrolwas selected as analogue source becauseit contains55% - 75% Cedrol.

Cedarwood Himalayan oil(target) contains besides Cedrol, various other constituents in low concentrations that are present in variable ranges, as shown in data matrix 1. None of the other constituents are registered under REACH and no reliable toxicity information is available.

 

Structural similarities and differences:

Cedarwood Himalayan oil(target) as well asCedarwood Texas oil distilled - Cedrol (source)contain mostly Cedrol. Cedrol is a cyclic terpenoid and containsa tertiary alcohol group. The remaining constituents ofCedarwood Himalayan oil(target) andCedarwood Texas oil distilled - Cedrol (source) differ. The remaining constituents in both source and target substance are varying in structure however all constituents are hydrocarbon compounds and some contain tertiary alcohol groups.

 

Absorption:

All constituents of the source and target substance are expected to be absorbed via the oral route based on their physico-chemical properties and molecular weights. The log kow values are comparable, ranges from 4.3 to 5.49 (KOWWIN 1.68) for the source, and was measured to be 5.5 for the target. Due to the LogKow values >4 Absorption though micellular solubilisation is expected to be an important mechanism of absorption.

 

Toxicodynamics

Both target and source contain Cedrol as main constituent, therefore the toxicodynamics for both UVCBs are expected to be comparable. No toxicokinetic information is available for the remaining, minor constituents inCedarwood Himalayan oil(target).

Conclusions on the toxicity endpoints and auto flammability:

Final hazard conclusion:

ForCedarwood Himalayan oil, a UVCB, the potential for mammalian toxicity hazards was derived fromCedarwood Texas oil distilled – Cedrol.Cedarwood Texas oil distilled – Cedrolis classified as a skin irritant (Skin Irrit. 2, H315) and a skin sensitiser (Skin Sens. 1B, H317)

The final conclusion on the classification ofCedarwood Himalayan oil is thereforeskin irritating (Skin Irrit. 2, H315) and a skin sensitiser (Skin Sens. 1B, H317).

 

Data matrix 1 for the comparison of the constituents inCedarwood Himalayan oil(target) andCedarwood Texas oil distilled - Cedrol(source)

NAME

CAS

Target

Target

Source

Source

 

 

Min.

Max.

Min.

Max.

Cedrol

77-53-2

50.00%

70.00%

>=55.00%

<=75.00%

 

 

 

 

 

 

rel-(1R,4S,4aR,8aR)-1,6-dimethyl-4-(propan-2-yl)-1,2,3,4,4a,7,8,8a-octahydronaphthalen-1-ol (Hydrolized Cadinene 1)

65700-78-9

1.00%

8.00%

-

-

rel-(1R,4S,4aR,8aS)-4,7-dimethyl-1-(propan-2-yl)-1,3,4,5,6,8a-hexahydronaphthalen-4a(2H)-ol

(Hydrolized Cadinene 2)

86023-67-8

1.00%

5.00%

-

-

rel-(1R,4R,4aR,8aS)-4,7-dimethyl-1-(propan-2-yl)-1,3,4,5,6,8a-hexahydronaphthalen-4a(2H)-ol

(Hydrolized Cadinene 3)

159990-04-2

0.50%

4.00%

-

-

2,5-dimethyl-8-(propan-2-yl)-1,2,3,4,6,7,8,8aoctahydronaphthalen-2-ol

(Hydrolized Cadinene 4)

57440-66-1

0.00%

3.00%

-

-

rel-(3R,3aR,5S,6R,7aR)-3,6,7,7-tetramethyloctahydro-3a,6-ethanoinden-5-ol

159517-24-5

1.00%

8.00%

-

-

(4beta)-cedr-8(15)-en-4-ol

114674-10-1

1.00%

7.00%

-

-

rel-(1R,2R,4aS,8aR)-4a-methyl-8-methylidene-2-(propan-2-yl)decahydronaphthalen-1-ol

38108-86-0

1.00%

5.00%

-

-

rel-(4aR,9S,9aS)-3,5,5,9-tetramethyl-2,4a,5,6,7,8,9,9a-octahydro-1H-benzo[7]annulen-9-ol

126999-77-7

1.00%

7.00%

-

-

rel-2-[(1R,4R,5R)-4,8-dimethylspiro[4.5]dec-7-en-1-yl]propan-2-ol

955007-43-9

1.00%

10.00%

-

-

beta-eudesmol

3287-59-0

0.50%

4.00%

-

-

(7S,9aS)-4,4,7,9a-tetramethyl-1,2,3,6,8,9-hexahydrobenzo[7]annulen-7-ol (Widdrol)

6892-80-4

-

-

>=2.00%

<8.00%

(1R, 5S, 7R)-2,6,6,8-tetramethyltricycle[5.3.1.0 1,5]undec-8-ene (Diepi-alpha-cedrene (Alpha-funebrene))

50894-66-1

-

-

>= 0.01%

<=2.00%

(6R)-1,5,5,9-tetramethylspiro[5.5]undeca-1,8-diene (Chamigrene alpha)

11912-83-5

-

-

>=0.01%

<=2.00%

(4aS,9aR)-3,5,5,9-tetramethyl-2,4a,5,6,7,9a-hexahydro-1H-benzo[7]annulene (Himachalene Gamma)

53111-25-4

-

-

>=0.01%

<=2.00%

3,5,5,9-tetramethyl-1,2,4a,6,7,8-hexahydrobenzo[7]annulene (Beta-himachalene)

1461-03-6

-

-

>=0.01%

<=2.00%

3,5,5-trimethyl-9-methylidene-2,4a,5,6,7,8,9,9a-octahydro-1H-benzo[7]annulene (Himachalene alpha)

3853-83-6

-

-

>=0.01%

<=2.00%

[1aS-(1aα,4aβ,8aR*)]-1,1a,4,4a,5,6,7,8-octahydro-2,4a,8,8-tetramethylcyclopropa[d]naphthalene (Thujopsene)

470-40-6

-

-

>=1.00%

<=5.00%

(R)-(+)-p-(1,2,2-trimethylcyclopentyl)toluene (Cuparene)

16982-00-6

-

-

>=0.01%

<=4.00%

(1S,2R,5S,7R)-2,6,6-trimethyl-8-methylidenetricyclo[5.3.1.0(1,5)]undecane (Beta-cedrene)

546-28-1

-

-

>=1.00%

<=5.00%

[3R-(3a,3ab,7b,8aa)]-2,3,4,7,8,8a-hexahydro-3,6,8,8-tetramethyl-1H-3a,7-methanoazulene (Cedrene alpha)

469-61-4

-

-

>=1.00%

<=5.00%

(2,6-dimethyl-6-(4-methyl-3-pentenyl)bicyclo[3.1.1]hept-2-ene)(

17699-05-7

-

-

>=0.01%

<=1.00%

Minor and unknown constituents

-

4.00%

15.00%

>=8.00%

<18.00%

 

Data matrix 2. Cedarwood Himalayan oil (target) and Cedarwood Texas oil distilled - Cedrol (source) information to support the read across.

CHEMICAL NAME

Cedarwood Himalayan oil

Cedarwood Texas oil distilled - Cedrol

Molecular structure

N/A

N/A

Target

Source

CAS

91771-47-0

91722-61-6

REACH registration

To be registered (Annex VII)

Yes

Einecs

294-939-5

294-461-7

Molecular formula

N/A

N/A

Molecular weight

N/A

N/A

Physico-chemical properties

 

Appearances

Brown, viscous liquid (IFF, 2017)

Light yellow solid mass

Melting point (˚C)

75.4(IFF, 2017)

- 42.5 (glass transition temperature)

Boiling point (˚C)

305.8 (IFF, 2017)

292.4

Vapour pressure (Pa)

2.81 (IFF, 2017)

0.67 (QSAR)

Water solubility (mg/L)

12.3 (IFF, 2017)

>= 0.03 -< = 29.25 (QSAR)

Log Kow

5.5 (IFF, 2017)

>= 4.33 -<=6.94(QSAR)

Autoflammability (˚C)

Read across

260

Human health

 

Acute toxicity (oral)

Read across

LD50 >5000 mg/kg bw (similar to OECD TG 401)(RIFM, 1974)

Skin irritation/corrosion

Read across

Skin irritating (OECD TG 439)

Not skin corrosive (OECD TG 431)

(NCS Sesquiterpenes HC/Alc consortium, 2017)

Eye irritation/corrosion

Read across

Not eye irritating (OECD TG 438) (NCS Sesquiterpenes HC/Alc consortium, 2017)

Skin sensitisation

Read across

Skin Sensitising (OECD TG 429) (NCS Sesquiterpenes HC/Alc consortium, 2017)

Genetic toxicity (Ames)

Read across

Negative (OECD TG 471)(NCS Sesquiterpenes HC/Alc consortium, 2017)

 

Justification for classification or non-classification

Based on the available information the test substance needs to be classified for skin irritation (Skin Irrit. 2, H315) in accordance with the criteria outlined in EU CLP (EC no. 1272/2008 and its amendments).