Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vitro
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
the study does not need to be conducted because the substance is classified as skin corrosion (Category 1, 1A, 1B or 1C)
Justification for type of information:
The study does not need to be conducted because the substance is classified as skin corrosion (Category 1B/1C). The classification of the substance is based on the available data for the classification of the constituents of the substance according to Guidance on the Application of CLP Criteria – Guidance to regulation (EC) No 1272/2008 ECHA.
Cross-reference
Reason / purpose:
reference to other study
Reference
Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 April 2019 – 23 June 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
OECD Guideline 431 Adopted 29 July 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
EssentÍal oil of Summer Savory/batch No. SSVI.ES.sample
Date of manufacture: 08.01.2019
Expration date of the batch: 08.01.2021
UVCB substance;
Purity test date: 18.01.2019
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Three-dimensional RhE models are obtained commercially (epiCS® supplied by CellSystems®). Additionally, 2 killed Human skin model surfaces were treated (SkinEthic RHE® model) in the same manner in order to generate non-specific MTT reduction.
Source strain:
other: human
Justification for test system used:
According to Annex VII of REACH Regulation, if new test data is required those must be derived from in vitro methods only.
Human skin models (epiCS®, SkinEthic™) (OECD 431) are accepted in vitro test method to detect skin corrosion/irritation (Category 1 or 2) and/or the absence of effects (not classified under CLP).
Vehicle:
unchanged (no vehicle)
Control samples:
yes, concurrent negative control
yes, concurrent no treatment
other:  Four killed control tissue models (two by exposure time) were added to the study, which underwent the entire testing procedure to generate a non-specific MTT reduction control.
Amount/concentration applied:
Preliminary test
The direct interaction of MTT with the test item was checked by adding 50 µL of the test item to 1 mL of the solution of MTT at 1 mg/mL (same conditions as in the main test). The preparation turned into two phases, one being yellow and one being purple, after 1 hour of incubation between 37.1°C and 37.4°C, 5% CO2.
As two phases appeared with a coloured part, direct interaction with MTT could be masked.
That is why four killed control tissue models (two by exposition time) were added to the study, which underwent the entire testing procedure to generate a non-specific MTT reduction control.

Main test
50 µL of the test item as supplied on 0.6 cm² human skin model
50 µL for positive control 8N KOH– Fisher Scientific
50 µL for negative control of distilled water– ADL Prochilab.
Duration of treatment / exposure:
3 minutes and 1 hour for test material
3 minutes and 1 hour for negative and positive control
Duration of post-treatment incubation (if applicable):
During 3 minutes at room temperature
During 1 hour at 370 C ± 10 C, 5%CO2
Number of replicates:
Two tissue replicates.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minutes and 1 hour after the test item application
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Corrosion
Other effects / acceptance of results:
This study is classified as acceptable and satisfies the requirement for Test Guideline OECD 431 for in vitro skin corrosion: Reconstructed human epidermis (RhE) test method.

Acceptability criteria:
The difference of viability between the 2 tissues treated with the negative control during 3 minutes was higher than 30% (40.9%).
Considering the results obtained (both epidermises have a viability much higher than 50% and are clearly classified as “Non-corrosive”), this deviation is considered as without impact on the conclusion of the study.

ASSESSMENT OF THE SKIN CORROSION

INDIVIDUAL AND AVERAGE VALUES AFTER 3 MINUTES EXPOSURE

 

TEST ITEM: Summer Savory oil

 

Application: 50 μL of the test item as supplied on 0.6 cm² human skin model

Application date: 16 May 2019

 

Table 1

 

Well ID

OD

Mean OD /

disc (#)

Mean OD /

product

 

Viability,

%

 

Mean

Viability,

%

 

Viability difference

between replicates, %

 

Negative control

 

SPL 1

 

 

 

SPL 2

0.716

0.704

0.719

 

0.456

0.461

0.494

 

0.713

 

 

 

0.471

 

 

 

 

0.592

 

120.44

 

 

 

79.56

 

 

 

100.00

 

 

 

40.9

Positive

control

 

SPL 3

 

 

 

SPL 4

 

0.100

0.116

0.131

 

0.106

0.110

0.115

 

0.116

 

 

 

0.111

 

 

 

0.114

 

19.59

 

 

 

18.75

 

 

 

19.17

 

 

 

0.8

Test item

PH-19/0208

 

SPL7

 

 

 

SPL8

0.240

0.241

0.245

 

0.190

0.218

0.220

 

0.242

 

 

 

0.210

 

 

 

0.0226

 

40.88

 

 

 

35.47

 

 

 

38.18

 

 

 

5.4

Test item

PH-19/0208

NSMTT

 

SPL7

 

 

 

SPL8

 

0.084

0.089

0.094

 

0.088

0.091

0.087

 

0.089

 

 

 

0.089

 

 

 

0.089

 

15.03

 

 

 

15.03

 

 

 

15.03

 

 

 

0.0

Test item PH-19/0208

corrected

 

 

 

 

 

 

23.14

 

Note

#: mean of 3 values

OD: optical density

SPL: sample

 

Acceptability criteria:

 

- Negative control: mean OD of the tissue replicates should be ≥ 0.8 and ≤ 2.8 for epiCS® model, for every exposure time. As the extract was diluted at 1:3 just before the OD measure, the acceptability criteria should be in the range ≥ 0.3 and ≤ 0.9 for the negative control.

- Positive control: mean viability of the tissue replicates exposed for 1 hour, expressed as % of the negative control, should be < 20% for epiCS model.

- Test item: in the range 20-100% viability, and for ODs ≥ 0.3, difference of viability between the two tissue replicates should not exceed 30%.

 

ASSESSMENT OF THE SKIN CORROSION

INDIVIDUAL AND AVERAGE VALUES AFTER 1 HOUR EXPOSURE

TEST ITEM: Summer Savory oil

 

Application: 50 µL of the test item as supplied on 0.6 cm² human skin model

Application date: 16 May 2019

Table 2

 

Well ID

OD

Mean OD /

disc (#)

Mean OD /

product

 

Viability,

%

 

Mean

Viability,

%

 

Viability difference

between replicates, %

 

Negative control

 

SPL 11

 

 

 

SPL 12

0.667

0.832

0.807

 

0.653

0.664

0.730

 

0.769

 

 

 

0.683

 

 

 

0.726

 

105.92

 

 

 

94.08

 

 

 

100.00

 

 

 

11.8

Positive control

 

SPL 13

 

 

 

SPL 14

0.002

0.000

0.001

 

0.005

0.005

0.005

 

0.001

 

 

 

0.005

 

 

 

0.003

 

0.14

 

 

 

0.69

 

 

 

0.41

 

 

 

0.6

Test item

PH-19/0208

 

SPL 17

 

 

 

SPL 18

0.194

0.204

0.207

 

0.187

0.191

0.195

 

0.202

 

 

 

0.191

 

 

 

0.197

 

27.82

 

 

 

26.31

 

 

 

27.07

 

 

 

1.5

Test item

PH-189/0208

NSMTT

 

SPL 19

 

 

 

SPL 20

0.063

0.059

0.062

 

0.061

0.056

0.062

 

0.062

 

 

 

0.060

 

 

 

0.061

 

8.54

 

 

 

8.26

 

 

 

8.40

 

 

 

0.3

Test item

PH-19/0208

corrected

 

 

 

 

 

 

18.66

 

 

Note

#: mean of 3 values

OD: optical density

SPL: sample

 

Acceptability criteria:

- Negative control: mean OD of the tissue replicates should be ≥ 0.8 and ≤ 2.8 for epiCS® model, for every exposure time. As the extract was diluted at 1:3 just before the OD measure, the acceptability criteria should be in the range ≥ 0.3 and ≤ 0.9 for the negative control.

- Positive control: mean viability of the tissue replicates exposed for 1 hour, expressed as % of the negative control, should be < 20% for epiCS model.

- Test item: in the range 20-100% viability, and for ODs ≥ 0.3, difference of viability between the two tissue replicates should not exceed 30%.

ASSESSMENT OF THE SKIN CORROSION

CONCLUSION AFTER 3 MINUTES AND AFTER 1 HOUR EXPOSURE

 

TEST ITEM: Summer Savory oil

 

Application: 50 μL of the test item as supplied on 0.6 cm² human skin model

Application date: 16 May 2019

 

Table 3

 

Mean viability (%)

 

3 min exposure

1 hour exposure

Conclusion

Positive control

19.17

0.41

Corrosive sub-category 1B/1C

Test item

PH-19/0208

23.14

18.66

Corrosive sub-category 1B/1C

Note

 

The item is to be classified as non-corrosive:

-If the viability after 3 minutes’ exposure is greater than or equal to 50% and the viability after 1-hour exposure is greater than or equal to 15 %;

 

The item is to be classified as corrosive:

- If the viability after 3 minutes’ exposure is strictly less than 50%, or

- If the viability after 3 minutes’ exposure is greater than or equal to 50% and the viability after 1-hour exposure is strictly less than 15 %

 

Summary of Negative Control data after a treatment of 3 minutes

Date

Negative control

item

Replicate num1ber

OD

Mean OD

Viability (%)

Viability difference

between replicates (%)

10/01/2018

Distilled water

1

 

2

0.578

 

0.562

 

0.570

101.4

 

98.6

 

2.8

24/01/2018

Distilled water

1

 

2

0.710

 

0.750

 

0.730

97.26

 

102.74

 

5.5

21/02/2018

Distilled water

1

 

2

0.686

 

0.695

 

0.691

99.35

 

100.65

 

1.3

21/03/2018

Distilled water

1

 

2

0.667

 

0.805

 

0.736

90.63

 

109.38

 

18.8

05/04/2018

Distilled water

1

 

2

0.655

 

0.637

 

0.646

101.39

 

98.61

 

2.8

11/04/2018

Distilled water

1

 

2

0.787

 

0.643

 

0.715

110.07

 

89.93

 

20.1

23/05/2018

Distilled water

1

 

2

0.821

 

0.669

 

0.745

110.2

 

89.80

 

20.4

04/07/2018

Distilled water

1

 

2

0.649

 

0.641

 

0.645

100.62

 

99.38

 

1.2

08/08/2018

Distilled water

1

 

2

0.476

 

0.464

 

0.470

101.28

 

98.72

 

2.6

05/09/2018

Distilled water

1

 

2

0.570

 

0.727

 

0.649

87.90

 

112.10

 

24.2

10/10/2018

Distilled water

1

 

2

0.629

 

0.616

 

0.623

101.04

 

98.96

 

2.1

17/10/2018

Distilled water

1

 

2

0.612

 

0.635

 

0.624

98.16

 

101.84

 

3.70

05/12/2018

Distilled water

1

 

2

0.722

 

0.636

 

0.679

106.33

 

93.67

 

12.70

OD: optical density

Negative control item = Dulbecco's phosphate-buffered saline (DPBS) or distilled water.

The optical density was measured after a dilution of the extractions in isopropanol (1:3).

Summary of Negative Control data after a treatment of 1 hour

Date

Negative control

item

Replicate number

OD

Mean OD

Viability (%)

Viability difference

between replicates (%)

10/01/2018

Distilled water

1

 

2

0.772

 

0.604

 

0.688

112.21

 

87.79

 

24.4

24/01/2018

Distilled water

1

 

2

0.793

 

0.652

 

0.723

109.76

 

90.24

 

19.5

05/12/2018

Distilled water

7

 

8

0.655

 

0.668

 

0.662

99.02

 

100.98

 

2.0

21/03/2018

Distilled water

7

 

8

0.583

 

0.563

 

0.573

101.75

 

98.25

 

3.5

05/04/2018

Distilled water

9

 

10

0.640

 

0.656

 

0.648

98.77

 

101.23

 

2.5

11/04/2018

Distilled water

9

 

10

0.662

 

0.765

 

0.714

92.78

 

107.22

 

14.4

23/05/2018

Distilled water

9

 

10

0.713

 

0.830

 

0.772

92.42

 

107.58

 

15.2

04/07/2018

Distilled water

11

 

12

0.679

 

0.682

 

0.681

99.78

 

100.22

 

0.4

08/08/2018

Distilled water

15

 

16

0.455

 

0.457

 

0.456

99.78

 

100.22

 

0.4

05/09/2018

Distilled water

13

 

14

0.694

 

0.676

 

0.685

101.31

 

98.69

 

2.6

10/10/2018

Distilled water

7

 

8

0.683

 

0.634

 

0.659

103.72

 

96.28

 

7.4

17/10/2018

Distilled water

9

 

10

0.719

 

0.764

 

0.742

96.97

 

103.03

 

6.1

OD: optical density

Negative control item = Dulbecco's phosphate-buffered saline (DPBS) or distilled water.

The optical density was measured after a dilution of the extractions in isopropanol (1:3).

Summary of Positive Control data after a treatment of 3 minutes

Date

Positive control

item

Replicate number

OD

Mean OD

Viability (%)

Viability difference

between replicates (%)

10/01/2018

KOH 8N

3

 

4

0.046

 

0.185

 

0.116

8.07

 

32.46

 

24.4

24/01/2018

KOH 8N

3

 

4

0.562

 

0.420

 

0.491

76.99

 

57.53

 

19.5

21/02/2018

KOH 8N

3

 

4

0.025

 

0.059

 

0.042

3.62

 

8.54

 

4.9

21/03/2018

KOH 8N

3

 

4

0.048

 

0.033

 

0.041

6.52

 

4.48

 

2.0

05/04/2018

KOH 8N

3

 

4

0.030

 

0.036

 

0.033

4.64

 

5.57

 

0.9

11/04/2018

KOH 8N

3

 

4

0.034

 

0.041

 

0.038

4.76

 

5.73

 

1.0

23/05/2018

KOH 8N

3

 

4

0.144

 

0.244

 

0.194

19.33

 

32.75

 

13.4

04/07/2018

KOH 8N

3

 

4

0.044

 

0.041

 

0.043

6.82

 

6.36

 

0.5

08/08/2018

KOH 8N

3

 

4

0.035

 

0.042

 

0.039

7.45

 

8.94

 

1.5

05/09/2018

KOH 8N

3

 

4

0.001

 

0.003

 

0.002

0.15

 

0.46

 

0.3

10/10/2018

KOH 8N

3

 

4

0.260

 

0.154

 

0.207

41.77

 

24.74

 

17.0

17/10/2018

KOH 8N

3

 

4

0.055

 

0.054

 

0.055

8.82

 

8.66

 

0.2

05/12/2018

KOH 8N

3

 

4

0.050

 

0.226

 

0.138

7.36

 

33.28

 

25.9

OD: optical density

Positive control item = 8N potassium hydroxide.

The optical density was measured after a dilution of the extractions in isopropanol (1:3).

Summary of Positive Control data after a treatment of 1 hour

Date

Positive control

item

Replicate number

OD

Mean OD

Viability (%)

Viability difference

between replicates (%)

10/01/2018

KOH 8N

3

 

4

0.001

 

-0.001

 

0.0001

0.15

 

-0.15

 

0.3

24/01/2018

KOH 8N

3

 

4

0.009

 

0.008

 

0.009

1.25

 

1.11

 

0.1

21/02/2018

KOH 8N

17

 

18

0.004

 

0.002

 

0.003

0.52

 

0.26

 

0.3

21/03/2018

KOH 8N

9

 

10

0.001

 

0.002

 

0.002

0.17

 

0.35

 

0.2

05/04/2018

KOH 8N

11

 

12

0.003

 

0.004

 

0.004

0.46

 

0.62

 

0.2

11/04/2018

KOH 8N

11

 

12

0.002

 

0.004

 

0.003

0.28

 

0.56

 

0.3

23/05/2018

KOH 8N

11

 

12

0.003

 

0.003

 

0.003

0.39

 

0.39

 

0.0

04/07/2018

KOH 8N

13

 

14

0.009

 

0.015

 

0.012

1.32

 

2.20

 

0.9

08/08/2018

KOH 8N

17

 

18

0.003

 

0.003

 

0.003

0.66

 

0.66

 

0.0

05/09/2018

KOH 8N

15

 

16

0.007

 

0.005

 

0.006

1.02

 

0.73

 

0.3

10/10/2018

KOH 8N

9

 

10

0.000

 

0.000

 

0.000

0.00

 

0.00

 

0.0

17/10/2018

KOH 8N

11

 

12

0.002

 

0.001

 

0.002

0.27

 

0.13

 

0.10

05/12/2018

KOH 8N

9

 

10

0.001

 

0.002

 

0.002

0.15

 

0.3

 

0.20

OD: optical density

Positive control item = 8N potassium hydroxide.

The optical density was measured after a dilution of the extractions in isopropanol (1:3).

 

Additionally, 2 killed Human skin model surfaces were treated (SkinEthic RHE® model) in the same manner in order to generate non-specific MTT reduction.

 

The aim of the study was to evaluate the possible corrosive effects of the test item Summer Savory oil after topical administration on in vitro human reconstituted epidermis (epiCS®, CellSystems®).

The test item Summer Savory oil was applied as supplied, at the dose of 50 µL, to two tissue Human skin model surfaces (epiCS®, CellSystems®) during 3 minutes and 1 hour. The application was followed by a rinse with 20 mL of DPBS. 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.

Additionally, 2 killed Human skin model surfaces were treated (SkinEthic RHE® model) in the same manner in order to generate non-specific MTT reduction.

The experimental protocol was established in accordance with the OECD. Test Guideline No. 431 dated 29 July 2016 and the EU method Method B.40.bis. In vitro skin corrosion: Human skin model test of the Council Regulation No. 440/2008 dated 30 May 2008.

Three minutes and 1 hour after the test item application, the mean percent viability of the epidermis skins treated with the test item was 23.14% and 18.66%, versus 19.17% and 0.41%, respectively, with the positive control item (potassium hydroxide 8N).

 

In accordance with the Regulation (EC) No. 1272/2008, the results obtained under the test conditions enable to conclude that the test item Summer Savory oil has to be classified in Sub Category 1B/1C “Corrosive” according to the Regulation (EC)1272/2008.

The corresponding hazard statement H314: Causes severe skin burns and eye damage” has to be assigned.

Interpretation of results:
other: Corrosive sub-category 1B/1C
Conclusions:
In accordance with the Regulation (EC) No. 1272/2008, the results obtained under the test conditions enable to conclude that the test item Summer Savory oil has to be classified in Sub Category 1B/1C “Corrosive” according to the Regulation (EC)1272/2008.
The corresponding hazard statement is “H314: Causes severe skin burns and eye damage”.
Executive summary:

The aim of the study was to evaluate the possible corrosive effects of the test item Summer Savory oil after topical administration on in vitro human reconstituted epidermis (epiCS®, CellSystems®).

The test item Summer Savory oil was applied as supplied, at the dose of 50 µL, to two tissue Human skin model surfaces (epiCS®, CellSystems®) during 3 minutes and 1 hour. The application was followed by a rinse with 20 mL of DPBS. 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.

Additionally, 2 killed Human skin model surfaces were treated (SkinEthic RHE® model) in the same manner in order to generate non-specific MTT reduction.

 

The experimental protocol was established in accordance with the OECD. Test Guideline No. 431 dated 29 July 2016 and the EU method Method B.40.bis. In vitro skin corrosion: Human skin model test of the Council Regulation No. 440/2008 dated 30 May 2008.

 

3 minutes and 1 hour after the test item application, the mean percent viability of the epidermis skins treated with the test item was 23.14% and 18.66%, versus 19.17% and 0.41%, respectively, with the positive control item (potassium hydroxide 8N).

 

In accordance with the Regulation (EC) No. 1272/2008, the results obtained under the test conditions enable to conclude that the test item Summer Savory oil has to be classified in Sub Category 1B/1C “Corrosive” according to the Regulation (EC)1272/2008.

 

The corresponding hazard statement H314: Causes severe skin burns and eye damage” has to be assigned.

Data source

Referenceopen allclose all

Reference Type:
other company data
Title:
Unnamed
Year:
2018
Reference Type:
publication
Title:
Conjugated Dienes as Prohaptens in Contact Allergy: In Vivo and in Vitro Studies of Structure-Activity Relationships, Sensitizing Capacity, and Metabolic Activation
Author:
Moa Andresen Bergstrom, Kristina Luthman, J. Lars G. Nilsson, and Ann-Therese Karlberg,
Year:
2006
Bibliographic source:
Chem. Res. Toxicol. 2006, 19, 760-769

Materials and methods

Results and discussion

Applicant's summary and conclusion

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
The classification of the substance for skin sensitisation is based on the available data for the classification of the constituents of the substance. The classification was derived based on Section 3.4.3.3.1. of Annex I of Regulation (EC) No 1272/2008 and Section 3.4.3.3.1. of the Guidance on the Application of CLP Criteria – Guidance to regulation (EC) No 1272/2008 of ECHA, Version 5.0 –July 2017. Summer Savory oil shall be classified as a skin sensitiser, Category 1 according to Regulation (EC) No1272/2008 .
Executive summary:

The study does not need to be conducted because the substance is classified as skin corrosion (Category 1B/1C).

The classification of the substance for skin sensitisation is based on the available data for the classification of the constituents of the substance. The classification was derived based on Section 3.4.3.3.1. of Annex I of Regulation (EC) No 1272/2008 and Section 3.4.3.3.1. of the Guidance on the Application of CLP Criteria – Guidance to regulation (EC) No 1272/2008of ECHA, Version 5.0 –July 2017. Six of the known constituents (Dipentene, alpha-Terpinene, alpha-Pinene, beta–Pinene, beta-Bisabolene and beta-Caryophyllene) are classified as skin sensitiser, Category 1 or 1B, H317 (harmonized and self-classification). The maximum concentrations of each of the six constituents is above the CLP generic concentration limits ≥ 1% that triggers classification of the mixture as Skin sensitiser.

Therefore, the Summer Savory oil shall be classified as a skin sensitiser, Category 1 according to the criteria Regulation (EC) No1272/2008.