Registration Dossier

Administrative data

Description of key information

For skin irritation:

The mean percent tissue viability of the treated tissues was 106.1%, versus 1.3% in the positive control (5% Sodium Dodecyl Sulfate).

 

Under these experimental conditions and in accordance with the Regulation (CE) No.1272/2008, the test item was classified as non-irritant.

For eye irritation:

A component is present at a content superior to 10 % and is classified H319. Thereore the substance is by default classified H319 in accordance with the regulation (CE) N°1272/2008.

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Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 August 2017
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Study conducted according to OECD Guideline 439 with deviations: details of test system, volume of test item/negative/positive controls and historical data not reported
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
yes
Remarks:
details of test system, volume of test item/negative/positive controls and historical data not reported
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Robertet S.A. / 1928083
Test system:
human skin model
Source species:
human
Cell type:
other: Reconstructed Human Epidermidis (RHE) Model
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Reconstructed Human Epidermidis (RHE) Model

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature (RT, comprised between 18-24 °C)
- Temperature of post-treatment incubation: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Exposure to the test substance was followed by rinsing with phosphate buffer saline (PBS) and mechanically dried.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- Epidermis were then transferred to fresh medium and incubated at 37 °C for 42 additional hours.
- Cell viability is assessed by incubating the tissues for 3 hours with 0.3 mL MTT solution (1 mg/mL). The formazan crystals are extracted using 1.5 mL isopropanol for 2 hours at RT and quantified by spectrophotometry at 570 nm wavelength.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: For each treated tissue, the cell viability is expressed as the percentage of the mean negative control tissues.

NUMBER OF REPLICATE TISSUES:
- Three tissues replicates for test item, negative and positive controls

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if [the mean relative viability after 42 minutes exposure and 42 hours post incubation is ≤ 50% of the negative control].
- The test substance is considered to be non-irritant to skin [if the mean relative viability after 42 minutes exposure and 42 hours post incubation is > 50% of the negative control].
- A cut-off value of 50% viability of the negative control value was considered and used to classify test substances as irritant (I) or non-irritant (NI). The culture environment might allow the detection of very small quantities of cytokines secreted by the epidermis in response to topical application of test substances.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Test item was topically applied on three tissues replicates for 42 minutes at room temperature (RT, comprised between 18-24 °C).
Duration of treatment / exposure:
Test item was topically applied for 42 minutes at room temperature (RT, comprised between 18-24 °C).
Duration of post-treatment incubation (if applicable):
42 hours at 37 °C
Number of replicates:
Three tissues replicates for test item, negative and positive controls
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
42 minutes exposure
Value:
106.1
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:
TISSUE VIABILITY:
The mean percent tissue viability of the treated tissues was 106.1%, versus 1.3% in the positive control (5% Sodium Dodecyl Sulfate).

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes; The negative control data meet the acceptance criteria if the mean OD value of the 3 tissues is ≥ 1.2 at 570 nm. The Standard Deviation value is considered as valid if it is ≤ 18% according to the Performance Standards (ECVAM SIVS. 2007).
- Acceptance criteria met for positive control: Yes; The positive control data meet the acceptance criteria if the mean viability expressed as % of the NC is < 40 % and the Standard Deviation value is ≤ 18 %.
- Batch acceptance criteria: All test substance data from one batch are considered as valid if both the negative and the positive controls data fulfill the above criteria requirements.
According to these criteria the test is validated.

Table 7.3.1/1: Reconstructed Human Epidermis (RHE) model – results

OD 570 nm

Tissue 1

Tissue 2

Tissue 3

Mean

Mean

Tissue

Viability

(%)

Standard

Deviation

(%)

Conclusion

PBS

(Negative

Control)

1.850

1.905

1.904

2.186

2.264

2.277

2.240

2.261

2.309

2.133

100

10.0

Non

Irritant

SDS

(Positive

Control)

0.030

0.031

0.031

0.022

0.022

0.022

0.031

0.032

0.034

0.028

1.3

0.3

Irritant

Patchouli

Fraction

290737

2.259

2.256

2.270

2.264

2.307

2.336

2.232

2.231

2.209

2.263

106.1

1.8

Non

Irritant

Interpretation of results:
GHS criteria not met
Conclusions:
Under these experimental conditions and in accordance with the Regulation (CE) No.1272/2008, the test item was classified as non-irritant.
Executive summary:

An in vitro skin irritation study was performed according to OECD Guideline 439 and in compliance with GLP to evaluate the possible irritating effects of the test item after topical application on in vitro human reconstructed epidermis (SkinEthic RHE model).

 

The test item was applied on three tissues replicates of Reconstructed Human epidermis during 42 minutes, followed by a rinse with PBS and 42 hours post-incubation period at 37 °C. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was  quantitatively measured after extraction from tissues.

 

The mean percent tissue viabilities obtained with the negative control and positive controls were met the acceptance criteria and therefore validate the experiment.

 

The mean percent tissue viability of the treated tissues was 106.1%, versus 1.3% in the positive control (5% Sodium Dodecyl Sulfate).

 

Under these experimental conditions and in accordance with the Regulation (CE) No.1272/2008, the test item was classified as non-irritant.

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

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation, other
Remarks:
Classification based on calculation rules for mixtures of the CLP Regulation
Type of information:
calculation (if not (Q)SAR)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
accepted calculation method
Qualifier:
no guideline followed
Principles of method if other than guideline:
Classification based on calculation rules for mixtures of the CLP Regulation
Irritation parameter:
other:
Remarks on result:
other: Eye irritation Category 2
Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Conclusions:
A component (alpha-guaiene) is present at a content superior to 10 % and is classified H319. Thereore the substance is by default classified H319.
Executive summary:

The NCS is composed of several identified constituents and in that, it can be considered as a mixture according to the definition of the CLP Regulation. The decision logic for classification of mixtures from the ECHA Guidance on the Application of the CLP Criteria (2017) was used to determine the serious eye damage/ eye irritant hazard of the registered substance. The decision of classification as eye Damage category 2 was based on existing data on constituents (additivity principles) the registered substance has a constituent classified as Eye Damage Category 2 present at more than 10% and should be classified as a Eye damage category 2 without further testing according to the rules for classification of mixtures of Regulation (EC) No 1272/2008.

The component alpha-guaiene (EC N°641 -402 -1) is present at a content superior to 10 % and is classified H319 (IFRA elbelling manual). Thereore the substance is by default classified H319.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

For skin irritation:

The mean percent tissue viability of the treated tissues was 106.1%, versus 1.3% in the positive control (5% Sodium Dodecyl Sulfate).

 

Under these experimental conditions and in accordance with the Regulation (CE) No.1272/2008, the test item was classified as non-irritant.

For eye irritation:

A component is present at a content superior to 10 % and is classified H319. Thereore the substance is by default classified H319 in accordance with the regulation (CE) N°1272/2008.