Juniper, Juniperus mexicana, ext.

Endpoint summary

Description of key information

Skin irritation in vitro (OECD TG 439): skin irrit. 2 (read across from Cedrol, Cedarwood Texas oil distilled)

Skin corrosion in vitro (OECD TG 431): not classified (read across from Cedrol, Cedarwood Texas oil distilled)

Eye irritation in vitro (OECD TG 438): not classified (read across from Cedrol, Cedarwood Texas oil distilled)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

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 classified

Remarks:

based on CLP criteria

Conclusions:

Under the conditions of the test, the Cedrol, Cedarwood Texas oil distilled was not corrosive. Based on this result, the 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).

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.

Reference 2

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".
29 November 2017 READ-ACROSS / SKIN IRRITATION / CW-TX-CRUDE I&B9W8768R001F02


1 Executive Summary
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 as appropriated to predict the endpoint “Skin irritation” for the target substance “Cedarwood Texas oil, Crude” (CW-TX-Crude) because:

 An OECD-439 in vitro skin irritation study (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method; test guideline adopted in 2015) and an OECD-431 in vitro skin corrosion study (In vitro skin corrosion: reconstructed human epidermis, RHE, test guideline adopted in 2016) are available for the source substance “Cedarwood Texas oil, Cedrol” (CW-TX-Cedrol), which composition is very similar to CW-TX-Crude, notably regarding the constituents that may drive the skin irritation effects;
 The read-across is based on the comparison of the composition of two UVCBs that are very close one another, the raw material being the same, the process the same except an additional distillation step for the source substance, and the constituents the same with variations only in their concentrations;
 This read-across prediction intends to propose a classification, according to CLP Regulation EC/1272/2008 and its amendments, as a skin irritant category-2;
 Conclusions on CLP Classification and Risk Assessment comply with the REACh EC/1907/2006 and amendments regulatory requirements.

This report follows the RAAF method and so presents:
1) The hypothesis: analogue read-across approach based on the constituents, notably the ones related to the skin irritation/corrosion effects;
2) The scientific justifications (“Assessment Elements”) and their evaluation (“Assessment Options”), which underline the coherence of the relationship between the variations of the irritant constituents and the skin irritant properties of the whole UVCB;
3) The conclusions, which are summarised hereafter.

Reason / purpose for cross-reference:

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

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 classified

Remarks:

based on CLP criteria

Conclusions:

Based on the results obtained using Cedrol, Cedarwood Texas oil distilled skin corrosion information for read across. Cedarwood Texas crude oil 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).

Executive summary:

Cedarwood Texas crude oil is not corrosive to the skin, based on the results from the (OECDTG431) source study performed with Cedrol, Cedarwood Texas oil distilled. The justification for this read across is provided in the attached justification for type of information.

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.

Reference 3

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

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 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:

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

Conclusions:

Based on the results obtained, it can be concluded that Cedrol, Cedarwood Texas oil distilled needs to be classified for skin irritation (Skin Irrit. 2 / H315) in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).

Executive summary:

The skin irritation potential of Cedrol, Cedarwood Texas oil distilled 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.

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. Therefore the substance should be classified as category 1 according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations and according to the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).

Reference 4

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".
29 November 2017 READ-ACROSS / SKIN IRRITATION / CW-TX-CRUDE I&B9W8768R001F02

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 as appropriated to predict the endpoint “Skin irritation” for the target substance “Cedarwood Texas oil, Crude” (CW-TX-Crude) because:

An OECD-439 in vitro skin irritation study (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method; test guideline adopted in 2015) and an OECD-431 in vitro skin corrosion study (In vitro skin corrosion: reconstructed human epidermis, RHE, test guideline adopted in 2016) are available for the source substance “Cedarwood Texas oil, Cedrol” (CW-TX-Cedrol), which composition is very similar to CW-TX-Crude, notably regarding the constituents that may drive the skin irritation effects;
The read-across is based on the comparison of the composition of two UVCBs that are very close one another, the raw material being the same, the process the same except an additional distillation step for the source substance, and the constituents the same with variations only in their concentrations;
This read-across prediction intends to propose a classification, according to CLP Regulation EC/1272/2008 and its amendments, as a skin irritant category-2;
Conclusions on CLP Classification and Risk Assessment comply with the REACh EC/1907/2006 and amendments regulatory requirements.

This report follows the RAAF method and so presents:
1) The hypothesis: analogue read-across approach based on the constituents, notably the ones related to the skin irritation/corrosion effects;
2) The scientific justifications (“Assessment Elements”) and their evaluation (“Assessment Options”), which underline the coherence of the relationship between the variations of the irritant constituents and the skin irritant properties of the whole UVCB;
3) The conclusions, which are summarised hereafter.

Reason / purpose for cross-reference:

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

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:

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

Conclusions:

Based on the results obtained using Cedrol, Cedarwood Texas oil distilled skin irritation information for read across. Cedarwood Texas crude oil needs to be classified for skin irritation (Skin Irrit. 2 / H315), in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).

Executive summary:

Cedarwood Texas crude oil was identified as a skin irritant (Skin Irrit. 2 / H315) based on the results from the (OECDTG439) source study performed with Cedrol, Cedarwood Texas oil distilled. The justification for this read across is provided in the attached justification for type of information.

The skin irritation potential of Cedrol, Cedarwood Texas oil distilled 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.

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. Therefore the substance should be classified as category 1 according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations and according to the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

26 July, 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and 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 classified

Remarks:

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

Conclusions:

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

Executive summary:

The eye hazard potential of 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 does not need to be classified as eye irritant according to the classification criteria outlined in Annex I of 1272/2008/EC (CLP).

Reference 2

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".
29 November 2017 READ-ACROSS / EYE IRRITATION / CW-TX-CRUDE I&B9W8768R001F2.0

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 Crude (CW-TX-Crude) because:

An OECD-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) is available for the substance Cedarwood Texas Cedrol (CW-TX-Cedrol), which composition is very similar to CW-TX-Crude, notably regarding the constituents that may drive the eye irritant effects;
This read-across prediction proposes not to classify Cedarwood Texas Crude as eye irritant, according to CLP Regulation EC/1272/2008 and its amendments.
Conclusions on CLP Classification and Risk Assessment comply with the REACh EC/1907/2006 and amendments regulatory requirements.

This report follows the RAAF method and so presents:
1) The hypothesis: analogue read-across approach based on the constituents related to possible eye irritation effects;
2) The scientific justifications (“Assessment Elements”) and their evaluation (“Assessment Options”), which underline the coherence of the relationship between the variations of the irritant constituents and the eye irritant properties of the whole UVCB;
3) The conclusions, 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 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.

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 classified

Remarks:

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

Conclusions:

Based on the results obtained using Cedrol, Cedarwood Texas oil distilled eye irritation information for read across, Cedarwood Texas crude oil 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:

Cedarwood Texas crude oil is not irritant to the eye, based on the results from the (OECDTG437) source study performed with Cedrol, Cedarwood Texas oil distilled. The justification for this read across is provided in the attached justification for type of information.

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 does not need to be classified as eye irritant according to the classification criteria outlined in Annex I of 1272/2008/EC (CLP).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The skin and eye irritation for Cedarwood Texas crude oil is assessed by using read across from Cedrol, Cedarwood Texas oil distilled. The experimental skin irritation and corrosion information on Cedrol, Cedarwood Texas oil distilled is summarised and the read-across justification is attached to the respective target records.

Skin Irritation information for Cedrol, Cedarwood Texas oil distilled

The skin irritation potential of Cedrol, Cedarwood Texas oil distilled 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.

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 information for Cedrol, Cedarwood Texas oil distilled

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 information for Cedrol, Cedarwood Texas oil distilled

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.

Justification for classification or non-classification

Based on the available data, it can be concluded that Cedarwood Texas oil - Crude needs to be classified for skin irritation (Skin Irrit. 2 / H315) but not for eye irritation in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).